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The muscular control of eyecup movements in the crab Carcinus maenas has been studied by both extracellular and intracellular recording from the nine eyecup muscles. Each muscle involved in optokinetic movements is supplied by a fast and slow axon and each consists of a histologically mixed spectrum of fibres ranging from (Felderstruktur). Muscle 19a, which only participates in the withdrawal reflex consists of phasic fibres only. In general, a fast axon preferentially innervates the phasic fibres and a slow axon the tonic ones. During optokinetic movements the muscles are activated by a complex motor output programme, which is different, not merely the reverse for movements in opposite directions. Both tonic and phasic muscle fibres are active but the latter are only active at greater amplitudes of stimulus movement. Tonic activity is responsible from maintaining the eyecup position in space and for low velocity, small amplitude movements. Phasic activity is recruited during large amplitude movements and is also responsible for fast movement and eyecup tremor. The protective withdrawal reflex overrides any other eyecup movement and involves the firing of two axons in the optic tract, one supplying a group of two, the other a group of three muscles. One of the muscles involved in this eyecup withdrawal movement away from the mid-line is also active during horizontal optokinetic movements of the eyecup towards the mid-line. It is suggested that interpretation of eyecup muscle activity is more intelligible if the whole group of muscles, rather than the individual muscles themselves, is regarded as the functional unit.

1. Physiological experiments were devised to study the electrical responses of ochinoderm muscle during contraction. The smooth muscle forming the lantern retractors of Eching conducts spike potentials at a speed off 4 cms. A sec. 2. Three muscle systems examined with the electron microscope reveal a layout that varies in complexity. The muscle cells give rise to processes that are synapsed upon by nerves. 3. The area containing the ribbon axons described by Smith (1950) was examined and the ribbon axons shown to be selectively stained muscle cells. 4. In the three muscle systems examined controlling ganglion was found to be associated sytams. 5. The ganglia contained large numbers of structures that are described as synapses. These synapses are of a simple type and do not possess the specialisations that are present in the synapses of some other invertebrate phyla. 6. The sensory cells of the epithalium are described and an examination has indicated that the cells of the general epithelial are all sensory and contribute axons to the nerve plexus. Some of these epithalial cells are modified into probable chemoreceptors, light receptors and are found in statocysts. 7. A hypothesis to account for the co-ordination of the behaviour of echinoderms in propounded.

CHAPTER 1 A brief introduction is given to the structure, biochemistry and electrophysiological and mechanical properties of fish muscle. CHAPTER 2 1. The neuromuscular end plates and preterminal axons of cod, Gadus morhua, fast myotomal muscle were stained for cholinesterase activity. 2. The number of end plates per fibre on superficial fast fibres (17.88 +/- 2.13, mean +/- 1 S.D.) was significantly higher than that of deep fast fibres (14,79 +/- 2.48, P<< 0.001). A small degree of multi-terminal innervation was noted. The end plates showed a great variety in structure and size. 3. Fast muscle contains fibres with a wide range of diameters (20-240 μm). However, no correlation was found between the number of end plates per fibre and fibre diameter. CHAPTER 3 1. The force-velocity characteristics of threads of natural actomyosin, and purified component proteins, from dogfish fast and slow muscle, and rabbit fast skeletal and cardiac muscle have been investigated. 2. Maximum isometric tensions were around 30-70 g cm-2. The time taken to reach full tension after activation with ATP was 2-8 min. 3. Force-velocity curves obtained could be fitted to a linear form of Hill's equation (1938). In common with intact and skinned fibre studies, points below 0.7 P0 were found to lie on a straight line. 4. Maximum contraction velocities were around 10-2 Ls-1, 2-3 orders of magnitude lower than those of intact muscle fibres. 5. The relative velocities of the different thread types do not reflect those of the corresponding muscles, on the basis of measurements on intact fibres, and on measurements of actomyosin/myofibrillar ATPase activities. 6. It is concluded that filament formation, geometry and packing, and not differences in cross bridge cycling rates, largely determine the observed properties of actomyosin threads. CHAPTER 4 A description of the apparatus used to study the isometric and isotonic properties of skinned fibres is given, together with the methods and protocol used in Chapters 5-7. CHAPTER 5 1. The pCa-tension relationship of cod, Gadus morhua, and dogfish, Scyliorhinus canicula, fast and slow skinned fibres isolated from the myotomal muscles was investigated. 2. Maximum isometric tensions were 1.9 +/- 0.12 (mean +/- 1 S.E.) (fast) and 0.85 +/- 0.10 (slow) for cod fibres, and 1.87 +/- 0.09 (fast) and 0.84 +/- 0.04 (slow) for dogfish 3. Sigmoid pCa-tension curves were obtained for all fibre types. Values for the half maximally activating [Ca2+], and n, the minimum number of Ca2+ binding sites involved in activation, were calculated: Thus, the minimum number of Ca2+ binding sites in cod is two, in dogfish, four. In both fish, greater cooperativity is exhibited by the fast muscle. CHAPTER 6 1. Force-velocity curves were derived from fast and slow skinned fibres isolated from cod and dogfish myotomal muscles. 2. The extrapolated V and the constants a and b were calculated max from a linear form of Hill's equation: 3. These results are discussed with reference to previous studies of the P-V relationship in amphibian and mammalian muscle. The relationship between a/P₀ and efficiency, and its bearing on the present results is discussed.4. Velocity transients showed a small departure from linearity in all experiments, with velocity decreasing continuously during release (usually < 25% over the first 250 ms after release). This is a feature common to many previous experiments on skinned and intact fibres. The decrease in velocity during release was particularly marked in cod slow fibres. Similar results have been reported in amphibian slow muscle. CHAPTER 7 1. Contraction velocity at low loads was studied in dogfish fast fibres during maximal and submaximal activations. 2. The velocity of contraction during the second 50 ms interval after the onset of release was reduced significantly at low [Ca2+], The shape of the velocity transient was found to be dependent on [Ca2+]. The rate of decrease of velocity was greater in submaximal than in maximal activations. 3. A brief review of previous studies on the dependence of Vmax on max [Ca2+] is given. 4. The results are discussed in the light of recent evidence for length dependent changes in the contractile system. Possible mechanisms for a Ca2+ and length dependent inactivation process are considered. CHAPTER 8 The major outstanding problems are stated, and suggestions are made for further work which may give a greater insight into the molecular events underlying contraction in fish muscle.

1. The sarcoplasmic reticulum from fish red and white muscle has been isolated and compared with that of mammalian sarcoplasmic reticulum (SR). The white muscle SR is shown to be similar to mammalian fast muscle with the majority of the protein being accounted for by a band with molecular weighty of approximately 100,000. This has been demonstrated in several species to correspond to the calcium pump protein. The red muscle SR pumps Ca2+ ions and hydrolyses ATP at approximately half the rate of the white SR. Gel electrophoresis of the SR vesicles shows that there is not sufficient 100,000 dalton protein in the red muscle SR to account for the relative rates of pumping. It is suggested that fish red muscle SR resembles mammalian slow fibres in that the pump protein's molecular weight is not the same as the white. Unlike mammalian SR, vesicles isolated from trout red muscle or not stimulated by commercially available cAMP dependent protein kinases. 2. The microsomal pellet of muscle is generally contaminated with mitochondrial fragments and sarcolemmal vesicles. Pure sarcoplasmic reticulum fractions have been obtained from plaice 2+ 2+ muscle and the characteristics of the Ca2+ ATPase and Ca2+ uptake studied. The protein composition of the vesicles, the dependence on Mg2+, Ca2+, K+ and ADP are demonstrated to be similar to that of rabbit muscle. A comparison of the levels of the phosphorylated enzyme intermediate with those obtained by other workers shows that the pump protein concentration is similar to rabbit fast skeletal muscle. 3. The effect of evolutionary temperature adaptation on the sarcoplasmic reticulum of different species of fish has been examined. The Ca2+ ATPase shows an inverse h correlation with the cell temperature of the species at O°C, and this difference is reflected in the relative rates of Ca2+ uptake. Activation enthalpy increases with increasing environmental temperature, while activation entropy goes from negative values in .the cold adapted to positive values in the hot adapted species. Unlike other enzyme systems, the activation energy shows no correlation with temperature. Acclimation to seasonal temperature changes has also been studied. Unlike adaptation on evolutionary time scales, acclimation does not affect the activation entropy of the ATPase, and there is no significant difference in the rates of ATP hydrolysis. It is concluded that the mechanisms of compensation are different in adaption to evolutionary and seasonal temperature changes. Evidence for acclimation to low temperatures involving an increase in the amount of sarcoplasmic reticulum is reviewed. 4. The enzyme-substrate affinity has been monitored by measuring the KCa of the Ca2+ ATPase at different temperatures for four different species of teleost. As with the Vmax estimations, compensatory changes are shown in the ATPase. KCa values, estimated at O°C are found to be inversely related Ca. to the animal's cell temperature. When estimated at that temperature, the values are similar. These results are discussed in relation to the observed prepondence of parvalbumins in fish muscle, and a possible role of the parvalbumins is considered. 5. In even the purest of SR preparations, i.e. those with no contaminating marker enzymes, a Ca2+ independent ATPase activity can be demonstrated. Unlike the Ca2+-ATPase the Ca2+ -independent ATPase activity is not related to habitat temperature, and the activation entropy is not proportional to cell temperature. As shown in the, rabbit, incubation of the SR in the presence of 0.1% Triton X-100 converts the basal ATPase activity in all except the lightest fractions to Ca2+ dependence. It is suggested that the Ca2+ independent ATPase represents a form of the pump protein uncoupled from Ca2+ transport, with the two forms of the ATPase in thermal equilibrium. Supportive evidence is provided for this theory. The ratio of Ca2+ dependent to total ATPase is temperature dependent, increasing to maximal value at and above the animal's cell temperature. For example, the ratio of approximately 0.25 at O°C in Tilapia mariae (a tropical species), increasing to 0.85 at 25°0. In contrast, the ratio for Myoxocephalus scorpius (North Sea) is only slightly affected by assay temperature and remains at about 0.95 throughout the range O - 33°C. The activation energy for the conversion has been estimated for two species and compared with that of rabbit. It is possible that the contribution of the activation energy for the conversion may account for the poor correlation of the DeltaG# of the Ca2+-dependent ATPase with environment temperature.

A. A study has been made of the tension responses and relative sliding movements of the actin and myosin filaments which result when controlled length changes of variable amplitude and velocity are applied to contracting frog's muscle. B. The tension increment produced by a 'ramp and hold' stretch of approximately 1mm (about 4 % of muscle length) consists of three phases whose limits are defined by two points, designated S1 and S2, where the slope of the increase of tension changes abruptly. S1 and S2 correspond with muscle extensions of 40-45 mum and 325 - 375mum, respectively. C. Displacement of the actin and myosin filaments was recorded simultaneously by monitoring changes in the diffraction spectra produced by illuminating a small area of muscle with a laser. The spectra were projected onto a screen and photographed with a cine-camera. The spacing of the 1st order lines was measured and used to calculate sarcomere length. D. Cine photography of diffraction spectra showed that a variable, and sometimes large, proportion of the length change applied to the muscle is taken up by extension of elastic elements arranged in series with the sarcomeres. During the early part of the stretch, filament displacement is less than would be anticipated if all of the external length change was distributed uniformly amongst the sarcomeres. E. The way in which sarcomeres shorten during the onset and development of an isometric tetanus shows that the series elastic elements become progressively stiffer with increasing extension; at peak tetanic tension their compliance is found to be approximately 6.5 x 10-4m.N-1. F. When the actin and myosin filaments are forcibly displaced by about 11-12nm from their 'steady state' position at the peak of an isometric tetanus, the sarcomere length increases abruptly by some 20- 30nm, a phenomenon referred to as rapid 'give'. The 11-12nm displacement before rapid 'give' occurs, represents the range of movement of the filaments over which the myosin cross-bridges can be distorted yet remain attached to the actin active site. This range of movement is found to be independent of sarcomere length, and hence, of interfilamentary spacing. Crossbridges are bridges are forcibly broken when the filaments are made to move by more than 11-12nm. The sudden increase in compliance of the sarcomeres which results effects an abrupt shortening of the stretched elastic elements (elastic recoil) at the expense of the sarcomeres in series with them, producing rapid 'give'. G. The stiffness of the sarcomeres and the force they are able to bear immediately prior to rapid 'give' (the maximum 'holding' force, designated Ps2) both directly proportional to filament overlap. Hence, the stiffness of an individual cross-bridge and its affinity for the actin active region are quantities which must be independent of the surface-to-surface separation of the filaments. The minimum stiffness of a single cross-bridge is estimated to be around 2.7 x 10-4 N.m-1. H. Tension changes and sarcomere movements during single and double cycles of stretch and release were recorded. The events occurring during a second cycle stretch differ markedly from those during a first. Cross-bridges can accommodate a wider range of filament movement without detaching , 18 nm as compared with 12 nm, if the second stretch is applied without delay after the proceeding release. The stiffness of the sarcomeres is approximately the same for a second cycle stretch ( 5.3 x 1012N.m-2 per metre extension of each half sarcomere) as for a first (5.95 x 1012N. 9 m-2 per metre extension of each half sarcomere), indicating that the number of cross-bridges holding the filaments together is not altered appreciably. These results are compatible with Huxley & Simmons recent model. They suggest an actin active region of length 15 nm, made up of four regularly spaced (~5.0 nm separation) attachment sites, corresponding with individual actin monomers.

The lateral musculature of elvers is differentiated into two fibre type on the basis of alkaline-labile (pH 10.2) myofibrillar ATPase activity. Slow muscle forms a relatively homogeneous fibre population, whereas fast muscle shows a heterogeneity with respect to both fibre size, and position in the myotome. The low aerobic capacity of slow fibres reflects the energetic requirement of anguilliform locomotion. A morphological continuum of myogenic cells occurs within mature; differentiated myotomal muscle similar to that described for embryonic myogenesis. No evidence could be found for regional growth nodes. Small fast fibres (< 100μm2) represent immature, but differentiated fibres undergoing hypertrophy. There is considerable variation in the capillary supply to both fast and slow muscle, and between homologous muscle off different species. Methods are described to determine the minimum sample size required for a stable, reproducible parameter estimate, and to assess the orientation of the capillary network. This is shown to be highly anisotropic. Capillary volume and surface densities are thought to be the most appropriated indices to use with fish muscle. The springs migration of elvers is shown to be a mixed population, of similar annual composition. The main migratory wave are true post-metamorphic, juvenile eels. There is a partial (Precht type 3) compensation in VO2 on acclimation to 10° and 29°C. The extent of the physiological acclimation reflects the environmental constraints of the migration. Differences in structure and complexity of multiple and focal innervation were investigated using fast muscle from representative teleosts. Endplate structure is similar in both types. Cod ventral spinal nerves have fewer motor, but more sensory axons than homologous nerves in eel. A novel way of visualizing the extent of inter- end intra segmental branching of nerves, intracellular marking of nerve routes with cobalt, reveals extensive branching in cod mytomes and cross-innervation between at least 3 segments. In cel, branching is restricted to a single mytome. These results reflect the mechanical and nervous control over the locomotory waveform.

The effect of ambient temperature on the ACh-induced tension response of the ABRM of Mytilus edulis was examined. The latter was found to have many temperature dependent variables, these included the latent period, the rate of tension development, maximum tension and the relaxation rate. Pmax was found to show a negative temperature coefficent. K+ contractures also showed a negative temperature co-efficent, Pmax approximately doubling for a 20°C decrease in temperature. The application of a cold shock during an ACh-induced contraction-relaxation cycle resulted in a transient increase in tension, the CIC. The production of a CTC was found to be dependent on the immediate presence of a stimulant, the time of application of the cold shock after the addition of stimulant, muscle length and temperature difference. As the temperature difference (ΔT) between the initial and cold shock solutions increased so the size of the CIC increased. The production of a CIC was found not to be directly related to the ACh or to the active state level yet it did not appear to be a passive phenomenon. A CIC was not produced when cold shock was applied to a muscle at rest or during catchy however, when catch was abolished by the application of relaxant a CIC could once again be elicited. Kinetic analysis of the CIC showed that, however, complex the mechanism two steps appear to be rate limiting, and the increase in tension with increasing AT was probably due to an increase in the availability of activator responsible for its production, rather than a differential effect on one of the rate limiting steps. Although it is conceivable that cold shock may exert a direct effect on the contractile proteins, evidence from the literature, and the experiments reported here, suggest that it is more likely that the CTC results from a transient increase in the level of myoplasmic Ca+2 Pharmacological investigation did not disprove this hypothesis. Of the possible sources of Ca+2 responsible for the CIC membrane associated sites seemed the most likely since under conditions which deplete this site no CIC was observed. Also the involvement of cAMP in the production of the CIC was largely excluded.

Water extract of the leaves of Erlangla cordifolia (S. Moore) - compositae (commonly known in Gikuyuland as Gathuna) has been traditionally used by the Gikuyu people of Kenya for centuries for the purpose of stimulating myometrial contractions in the process of parturition. The use of this extract has been called for when the progress of the birth process has been judged to be unsatisfactorily slow. The introduction to this work (Chapter 1) therefore surveys this use of the plant material and proceeds on to show the validity of using Erlangia material for the study of the process of muscle contraction. The section also includes a short discussion on the validity of the use of what could be termed a pathological condition - that is, the obstetric 'lazy' uterus - for the purpose of studying a normal physiological process (muscle contraction) at the cellular and molecular levels. Chapter 2 explains the methods used to obtain the crude extract from Erlangia leaves andvalso a purified compound, cordifene, from the same source, that was found by the author of this work to have stimulatory activity on contracting muscle. The chapter also deals with the methods used to characterize cordifene chemically. Chapter 3 describes the physiological experiments carried out to confirm the stimulatory activity of Erlangia extract and also of cordifene on the smooth muscle of the myometrium and that of the intestinal wall, in addition to similar effects on skeletal muscle. These experiments therefore confirmed the fact that the activity of Erlangia material has a common biochemical basis for all types of muscle at the molecular level. Stimulation of muscle contraction can be brought about through biochemical effects on the nerve(s) supplying the muscle or through direct effects on biochemical mechanisms occurring within the muscle cell itself. Chapter 4 of this work is therefore concerned with an investigation into the possibility of a chemical compound or compounds from Erlangia leaves that may be capable of influencing biochemical processes within the peripheral autonomic nervous system, as this system is known to be intricately involved in muscle contraction. Acetylcholine metabolism is important not only for the autonomic nervous system's biochemical role in muscle contraction: it is also important for the biochemical processes that take place in nervous impulse transmissions in general - with all the consequences that this has on all muscles, both voluntary and involuntary. For this reason, a possible effect of Erlangia material on the cholinesterase enzymes has been searched for Chapter 5 is concerned with a series of investigations into the different biochemical processes that occur in the muscle cell during the contractile activity and the mode of action by which Erlangia material might be influencing such processes. Chapter 6 is a discussion based on the results of the investigations reported in the previous sections. Erlangia material was found to react with ADP forming a complex and, besides probably inducing increased myosin ATPase activity, it was also found to induce marked configurational changes within the actomyosin molecule.

Chapter 1 General Introduction: The general introduction initially presents the major landmarks in muscle research of the last 3 millennia. The proteins of the contractile apparatus and their role in muscle contraction are described. There is then a description of how contractile proteins are known to alter through the expression of isoforms. Finally, a description of fish muscle and its fibre types is given, followed by the aims of the thesis. Chapter 2 Materials and Methods: An account is given of the materials and methods used throughout this thesis. This includes myofibril preparation and electrophoretic techniques. The techniques described are one and two dimensional polyacrylamide gel electrophoresis (PAGE), iso-electric focusing (lEF), peptide mapping, and one and two dimensional alkali-urea PAGE. The methods used to fix, stain and store the gels are then given, followed by the protocol used for Western blotting. Finally, the analysis of proteins bands is described. Chapter 3 Temperature and the plasticity of myofibrillar proteins during ontogeny in the Atlantic herring (Clupea harengus L.): Many aspects of development are influenced by temperature. The aim of this study was to investigate the effect of rearing temperature on the development and myofibrillar protein expression during ontogeny in the Atlantic herring (Clupea harengus L.). Chapter 4 The effect of body size on the myofibrillar protein composition of fast muscle fibres in the short horn sculpin (Myoxocephalus scorpius L.): The contractile properties of muscle vary with body length in fish. The aim of this study was determine if the proteins altered with body size in the short-horn sculpin (Myoxocephalus scorpius L.). Furthermore, could changes in protein expression be related to differences in contractile properties. Chapter 5 The myofibrillar proteins of Antarctic and sub-Antarctic fish: The myosins of Antarctic fish are specialised for function at low temperature. The aim of this study was to determine if the myofibrillar proteins present in Antarctic fish were highly conserved for function in this stable, low temperature environment. The variation in protein structure from myotomal fast muscles and the m. adductor profundis between five Antarctic fish species from two genera was compared with five sub-Antarctic species from four genera. The myofibrillar proteins of both the Antarctic and sub-Antarctic species showed a high degree of similarity between fish within the same genera. However, the isoforms present were considerably different between genera in both the Antarctic and sub-Antarctic species. Furthermore, the extent of the variation in protein isoforms between genera of the Antarctic fish was similar to that of the sub-Antarctic fish. This suggests that divergence in the tertiary structure of myosins from these species has occurred and that the Antarctic fish myofibrillar proteins are not highly conserved. Chapter 6 General Discussion: The major findings of the thesis are discussed in relation to the expression of myofibrillar proteins, with reference to further studies.