Neuromuscular Function and Disorders

future.

SYMBOLS, UNITS OF MEASUREMENT AND ABBREVIATIONS

Length

m = metre

cm = centimetre (10−2m)

mm = millimetre (10−3m)

μm = micrometre (10−6m)

Å = Ångstrom (10−10m)

Mass

g = gram

kg = kilogram

Time

s = second

ms = millisecond (10−3s)

μs = microsecond (10−6s)

Velocity

m/s = metres per second

Frequency

Hz = hertz (cycles per second)

Electrical

A = ampere

C = capacitance

E = e.m.f. (electromotive force)

I = current

Ω = ohm

R = resistance

V = volt

mV = millivolt (10−3V)

μV = microvolt (10−6V)

Chemical

mEq = milliequivalent

mM = millimolar

[ ] = concentation of

ACh = acetylcholine

AChE = acetylcholinesterase

AChR = acetylcholine receptor

ADP = adenosine diphosphate

AMP = adenosine monophosphate

ATP = adenosine triphosphate

BTX = bungarotoxin

Ca = calcium

ChE = cholinesterase

Cl = chlorine

CPK = creatine phosphokinase

CSF = cerebrospinal fluid

DFP = diisopropyl fluorophosphate

DNA = deoxyribonucleic acid

EDB = extensor digitorum brevis

EEG = electroencephalogram

EMG = electromyogram

EPP = end-plate potential

FHL = flexor hallucis longus

FSH = facioscapulohumeral (dystrophy)

Hg = mercury

HRP = horseradish peroxidase

K = potassium

LG = limbgirdle (dystrophy)

M(wave) = maximum evoked muscle response

m.e.p.p. = miniature end-plate potential

Na = sodium

PAS = periodic acid Schiff (stain)

RNA = ribonucleic acid

SMA = spinal muscular atrophy

SOL = soleus

STX = saxitonin

Statistical treatment

Unless otherwise stated, means have been expressed with standard deviations. Significances of differences between means have been estimated by the Student ‘t’ test.

PART 1

MUSCLE FIBRES AND MOTONEURONES

Outline

Chapter 1: THE MUSCLE FIBRE

Chapter 2: THE MOTONEURONE AND ITS AXON

Chapter 3: RESTING AND ACTION POTENTIALS

Chapter 4: THE NEUROMUSCULAR JUNCTION

Chapter 5: MUSCLE CONTRACTION

Chapter 6: MOTOR UNITS

Chapter 7: EXERCISE AND FATIGUE

Chapter 8: TROPHIC INTERACTIONS OF NERVE AND MUSCLE: DENERVATION AND REINNERVATION

Chapter 9: USE AND DISUSE

Chapter 10: TROPHIC INFLUENCE OF MUSCLE ON NERVE

Chapter 11: MUSCLE GROWTH

Chapter 12: AGEING

Chapter 1

THE MUSCLE FIBRE

Publisher Summary

This chapter discusses the structure of muscle fiber. Voluntary" muscles are composed of two types of muscle fibers. By far the most common are those fibers that make up nearly the entire bulk of the muscle and are described as extrafusal. This term distinguishes them from the much smaller muscle fibers that are found inside the muscle spindles. Because of their location, these smaller muscle fibers are referred to as intrafusal. At birth, human fibers are slender, measuring about 10–20 μm in diameter. Throughout childhood and particularly during the growth spurt in puberty, the sizes increase and eventually attain adult dimensions with most diameters in the 40–80 μm range. The membrane of the muscle fiber is termed the sarcolemma and is some 75 A thick. Just outside the sarcolemma and clearly visible in electronmicrographs is the basement membrane which is composed of proteins and polysaccharides. Each muscle fiber contains numerous nuclei which are dispersed along the inner surface of the sarcolemma, particularly in the region of the motor end-plate. The most obvious structures within the muscle fiber are the myofibrils, which are the units responsible for contraction and relaxation of the fiber.

The ‘voluntary’ muscles are composed of muscle fibres, of which two types are found. By far the commonest are those fibres which make up nearly all the bulk of the muscle and are described as extrafusal. This term distinguishes them from the much smaller muscle fibres which are found inside the muscle spindles; because of their location these last fibres are referred to as intrafusal. The special structure and function of the intrafusal fibres are of considerable interest and have been reviewed elsewhere (Matthews, 1972). This book will deal only with the extrafusal muscle fibres, however, for it is these fibres about which most is known in disease and it is their ineffectiveness which is ultimately responsible for the cardinal symptom of weakness.

NUMBERS AND SIZES OF MUSCLE FIBRES

Because of the time-consuming nature of the task involved, there are very few values for the numbers of extrafusal fibres in human voluntary muscles. On the basis of fibre counts in samples taken from cadaveric tissue, Feinstein et al. (1955) made the estimates set out in Table 1.1.

TABLE 1.1

Number of Muscle Fibres in Various Human Muscles (results given to nearest 50)

*Average values

The long gestation period in man allows sufficient time for the precursors of the muscle fibres to have finished dividing and to have formed the adult complement of fibres before birth (MacCallum, 1898). In species with a shorter gestation period the number of fibres continues to increase in the neonatal period. At birth the human fibres are slender, measuring about 10–20 μm in diameter. Throughout childhood, and particularly during the growth spurt in puberty, the sizes increase and eventually attain adult dimensions, with most diameters in the 40–80 μm range (see Figure 1 of Brooke and Engel, 1969). So far as length is concerned, it is assumed that individual fibres run from one end of the muscle to the other; in the human sartorius muscle such fibres may not be functionally continuous; certainly the thigh muscles possess more than one innervation zone and it is possible that long fibres may be composed of two or more shorter fibres arranged end-to-end.

Figure 1.1 (Upper) Structure of the sarcolemma, showing the double layer of lipid molecules and an ‘intrinsic’ protein (see text). (Lower) Membranes, nuclei and organelles seen at the surface of a muscle fibre; the mitochondrion would measure about 1.5 μm in its long diameter

STRUCTURE OF THE MUSCLE FIBRE

Sarcolemma (plasmalemma)

All living cells are bounded by a membrane; that of the muscle fibre is termed the sarcolemma thick. The sarcolemma resembles the membrane of the nerve fibre in possessing the special property of excitability, enabling electrical impulses to be transmitted down the length of the cell (see page 20). In the region of the innervation zone (motor end-plate) the sarcolemma is highly convoluted, due to the presence of junctional folds (page 27). Elsewhere along the surface of the fibre the membrane displays much shallower folds; these result from slackness of the membrane when the fibre is in its resting or contracted states and they disappear if the fibre is passively stretched. There are also numerous very small in-pocketings of membrane, the caveolae, which are connected to the surface membrane by narrow necks; their function is uncertain though they can also act as reserve sources of membrane during stretching of the fibre (Dulhunty and Franzini-Armstrong, 1975). Biochemical and ultrastructural investigations indicate that the sarcolemma is largely composed of lipid molecules arranged perpendicularly to the surface of the fibre and forming two layers (Figure 1.1, upper; see also Capaldi, 1974). The hydrophilic ‘heads’ of the lipid molecules form the internal and external surfaces of the membrane while the hydrophobic ‘tails’ make up the interior of the membrane. The membrane also contains proteins, of which two types are generally recognized; these are referred to as extrinsic and intrinsic. Extrinsic proteins are only attached at the internal or external surfaces of the membrane and can be dislodged relatively easily by chemical means. In contrast, intrinsic proteins penetrate the full thickness of the membrane and are difficult to remove. Among the special proteins known to be localized in the sarcolemma are (a) transport systems for sugars, lipids and, amino acids; (b) kinases for phosphorylating various membrane proteins; (c) adenylate cyclase, responsible for synthesizing cyclic AMP; (d) ATPase enzymes for pumping cations across the membrane (one type for sodium and potassium and another for calcium and magnesium); and (e) carrier molecules (ionophores), each of which can select one species of ion (sodium, potassium or chloride) and transfer it across the membrane (see page 24). In the end-plate region the sarcolemma also contains two special proteins which combine with acetylcholine; these are the acetylcholine receptor and the hydrolytic enzyme, acetylcholinesterase. Since much of the membrane lipid has a melting point below body-temperature the sarcolemma would be expected to have fluid properties. By labelling a spot of membrane with a fluorescent dye and then observing its enlargement under the microscope Fambrough et al. (1974) were able to show that this was indeed the case.

Basement membrane

Just outside the sarcolemma and clearly visible in electronmicrographs is the basement membrane (thick and is composed of proteins and polysaccharides. It is probable that the basement membrane provides a special ionic milieu for the muscle fibre by restricting the further diffusion of electrolytes once these have crossed the sarcolemma. In addition the basement membrane helps to maintain the shape of the muscle fibre by providing external support. If the muscle fibre is damaged the membrane may be spared and can then guide the regenerating myoblasts in the formation of a new fibre.

Nuclei

Each muscle fibre contains numerous nuclei (myonuclei) which, in health, are dispersed along the inner surface of the sarcolemma, particularly in the region of the motor end-plate. The nucleus is bounded by two membranes, the outer one of which may join the sarcoplasmic reticulum (Figure 1.1, lower).

Indistinguishable from the myonuclei with the light microscope are the nuclei of the satellite cells. These cells probably account for less than 1 per cent of the muscle fibre nuclei in the adult. They can only be differentiated from the myonuclei by the electronmicroscope, which reveals the presence of twin membranes separating the cytoplasm of the satellite cell from that of the muscle fibre. The satellite cells are of particular importance for the regeneration of muscle following disease or injury (page 100).

Myofibrils

The most obvious structures within the muscle fibre are the myofibrils, which are the units responsible for contraction and relaxation of the fibre (Figure 1.2). Each myofibril is about 1 μm in diameter and, even with the light microscope, can be seen to have a banded, or striated, appearance. The ‘dark’ and ‘light’ striations are termed the A- and I-bands respectively; in the relaxed muscle fibre the centre of the A-band has a light region, the H-zone, which is itself bisected by a ‘dark’ M-line. In the centre of the I-band is a dark Z-line (Z-disc). The striations are caused by the fact that the part of the myofibril in the A-band has a refractive index which is substantially higher than that of the fluid medium, or sarcoplasm, in which the myofibrils are embedded. The pattern of one dark band followed by one light band is repeated every 2.2 μm (measured with the muscle in the relaxed state), the segment between two successive Z-lines being termed a sarcomere. All the myofibrils within a healthy muscle fibre are in register, such that the light and dark bands of one myofibril are adjacent to the corresponding bands of neighbouring myofibrils, giving the whole muscle fibre a striped appearance. A satisfactory explanation for these muscle striations is now possible in terms of the disposition of the contractile macromolecules along the axis of the myofibril (page 35).

Figure 1.2 Part of the periphery of a muscle fibre. Of the several hundred myofibrils in the fibre the section shows only three (MF); their structure has been simplified and the foreground myofibril reflected slighty upwards. H, H-zone; M, M-line; Mi, mitochondrion; MN, myonucleus; SL, sarcolemma; SR, sarcoplasmic reticulum; T, transverse tubular system; Z, Z-line (see text)

Tubular systems

Early in the present century Veratti (1902) was able to stain a fine interlacing network within the muscle fibre (Figure 1.2). It was only many years later, with the aid of the electronmicroscope, that the details of this structure could be resolved into a tubular system comprising two parts (see, for example, Franzini-Armstrong and Porter, 1964).

The sarcoplasmic (endoplasmic) reticulum

An elaborate system of channels runs in the long axis of the muscle fibre and largely surrounds individual myofibrils. Through side branches the longitudinal channels enveloping one myofibril are connected to each other and to those around other myofibrils. When the muscle fibre contracts the longitudinal channels become shorter and wider.

The transverse tubular (T) system

This lies perpendicular to the long axis of the muscle fibre in the form of narrow zones of channels which encircle the myofibrils at regular intervals. In mammalian muscle fibres, including those of man, there are two zones of transverse tubules in each sarcomere; they lie at the junctions of the A- and I-bands. Interestingly, in cardiac muscle fibres and in the fibres of frogs, there is only one T-zone for each sarcomere and this is situated at the Z-line. As the T-tubules encircle the myofibrils they interrupt the longitudinal channels of the sarcoplasmic reticulum. At these points of contact the sarcoplasmic reticulum is somewhat dilated to form lateral sacs (or terminal cisterns); neighbouring sacs are connected together. Figure 1.2 shows that each of the relatively narrow T-tubules is embraced on either side by a lateral sac; the three elements are referred to as a triad. Electronmicroscopy reveals that the membranes of the T-tubules and sarcoplasmic reticulum, although closely apposed at the triads, remain intact. According to Peachey (1965) approximately 80 per cent of the transverse tubular system in a frog muscle fibre is surrounded by the sarcoplasmic reticulum. At the surface of the muscle fibre the T-tubules form small openings at the junctions of the A- and I-bands and their membranes become confluent with the sarcolemma. By examining muscle fibres bathed in solutions containing electron-dense material or fluorescent dyes, it has been shown that the T-tubules contain extracellular fluid. The principal function of the tubules is to conduct impulses from the surface to the interior of the muscle fibre. Each inwardly conducted impulse causes calcium ions to travel from the lateral sacs along the sarcoplasmic reticulum to the region of the myofibrils where the contraction will take place; afterwards the calcium ions are returned to the lateral sacs (page 40).

Sarcoplasmic components

Mitochondria

The mitochondria (sarcosomes) are ovoid structures, measuring some 1–2 μm in their longest diameters. They have a double membrane, of which the inner one is repeatedly folded to form cristae; these folds bulge into the central compartment of the mitochondria (Figure 1.1, lower). The mitochondria house the enzyme systems required for the Krebs tricarboxylic acid cycle which breaks down pyruvate to carbon dioxide and water. A large part of the energy released by the successive reactions is captured by a chain of cytochromes which enable ATP to be formed. There is now good evidence that the cytochrome chain is located on the membrane of the cristae. Mitochondria are found especially between the myofibrils in the region of the Z-line and also in relation to the nuclei and the motor end-plate. Although the mitochondria appear oval in longitudinal sections of muscle fibres, some are not ovoid but are much more complex; extremely bizarre shapes may occur in disease.

Ribosomes

in diameter which may occur singly or else in groups (polyribosomes). They are especially numerous in immature muscle fibres and in regenerating ones, being concerned with the synthesis of proteins; they are composed of RNA (ribonucleic acid).

Glycogen granules

in diameter, are scattered throughout the sarcoplasm and are the major sources of energy for the muscle fibre.

Lipid droplets

These are often to be found close to mitochondria and contain fatty acids or triglycerides; they form important supplementary sources of energy.

Chapter 2

THE MOTONEURONE AND ITS AXON

Publisher Summary

This chapter discusses motoneurons and their axons. When a muscle is required to contract it is sent the necessary instructions in the form of nerve impulses by large cells lying in the ventral grey matter of the spinal cord. These cells are the motoneurons, each of which consist of a cell body (soma, perikaryon) and special processes termed the dendrites and the axon. The soma contains the nucleus of the cell together with various structures. The function of the dendrites is to receive signals from other neurons while the major role of the axon is to transmit the resulting message to the muscle fibers. The lengths of the axons vary considerably. In the case of a man 180 cm tall, an axon running from the lumbosacral region of the spinal cord to one of the plantar muscles would be about 125 cm long. In contrast, the fibers passing in the cranial nerves to the external ocular muscles or to the muscles of the face or tongue would only measure about one-tenth of this length. The chapter discusses in detail, the various parts of the motoneurone.

When a muscle is required to contract it is sent the necessary instructions in the form of nerve impulses (action potentials) by large cells lying in the ventral grey matter of the spinal cord (or in a corresponding region of the brainstem). These cells are the motoneurones, each of which consists of a cell body (soma, perikaryon) and special processes termed the dendrites and the axon (Figure 2.1). The soma contains the nucleus of the cell together with various structures which will be considered later. The function of the dendrites is to receive signals from other neurones while the major role of the axon is to transmit a resulting message to the muscle fibres. The lengths of the axons vary considerably. In the case of a man 180 cm tall, an axon running from the lumbosacral region of the spinal cord to one of the plantar muscles would be about 125 cm long. In contrast, the fibres passing in the cranial nerves to the external ocular muscles, or to the muscles of the face or tongue, would only measure about one-tenth of this length. As the axon nears the muscle it begins to divide with each branch dividing further, so that eventually a single parent axon is able to make contact with many muscle fibres. The contact region between an axonal twig and a muscle fibre is termed a synapse, or neuromuscular junction. The synapse possess special structural and functional features which enable the impulse in the nerve fibre to be translated into an impulse in the muscle fibre through the action of a chemical link, the transmitter acetylcholine. In the normal adult mammal each muscle fibre has only one neuromuscular junction area and is innervated by only one motoneurone. Sherrington (1929) pointed out that a single motor nerve fibre and the colony of muscle fibres which it supplied could be considered as a functional entity, since each time the nerve fibre discharged an impulse the muscle fibres of the colony would be excited together; Sherrington described this entity as a motor unit (see Chapter 6).

Figure 2.1 (Upper) A motoneurone and the proximal part of its axon (see text). (Middle) A transverse section through the axon to show the arrangement of the myelin lamellae in relation to the axis cylinder and the cytoplasm of the Schwann cell. (Lower) Part of the axis cylinder, enlarged to show the neurotubules and neurofilaments

Many methods have been used to study the structure of the motoneurone. For example, Haggar and Barr (1950) examined serial sections of cat spinal cord and were able to make three-dimensional models of the soma and dendrites. Another technique, also using a light microscope, was devised by Chu (1954). Pieces of ventral horn were dissected from recent autopsy specimens of human spinal cord and were made into a crude suspension with physiological saline. Within this suspension a proportion of the motoneurones were completely dissociated from other tissue and yet retained considerable lengths of axon and dendrites suitable for examination. More recently a technique for injecting the dye procion yellow through a micropipette into the soma of a neurone has been applied to motoneurones; the dye diffuses into the dendrites and proximal axon, enabling the full extent of the cell to be visualized (Kellerth, 1973). For information concerning the fine structure of the motoneurone it has been necessary to turn to the electronmiscroscope and several comprehensive reports are now available (see, for example, Bodian, 1964). The various parts of the motoneurone will now be considered in more detail.

Soma

Within the ventral grey matter of the spinal cord, the cell bodies of the motoneurones are arranged in columns parallel to the long axis of the cord. The cells supplying the extrafusal muscle fibres are termed alpha-motoneurones in order to distinguish them from the gamma-motoneurones (fusi-motoneurones) innervating the small fibres within the muscle spindles. The cell bodies of the alpha-motoneurones are much larger than those of the gamma type and may have diameters of 100 μm; an average value would be about 70 μm.

The most prominent feature of the soma is the relatively large nucleus which is bounded by a wavy double membrane and contains nucleoplasm and a nucleolus. Within the nucleoplasm are the chromosomes, themselves made up of many genes, each of which consists of DNA. It is the genes which are ultimately responsible for directing the metabolic activities of the neurone. They do so by sending instructions, in the form of messenger RNA, out into the cytoplasm of the cell, possibly through the pores in the nuclear membrane which can be seen with the electronmicroscope. Since human motoneurones do not multiply after birth the chromosomes cannot be distinguished in the nucleoplasm of the motoneurone; all that can be seen in the latter are irregularly distributed particles, 10–20 μm in diameter. Within the nucleus the nucleolus appears as a large structure, measuring about 4 μm across and consisting mostly of ribosomal RNA. This RNA is made on the DNA templates of the genes and is eventually passed out from the nucleolus into the cytoplasm to take part in the synthesis of proteins.

Cytoplasm

The cytoplasm of the motoneurone soma contains several different types of organelle, the most prominent of which is the Nissl body. These bodies range from 0.5 to 3 μm in diameter and electronmicrographs show them to be composed of tightly packed arrays of endoplasmic reticulum. The membranes of the reticulum are studded with ribosomes, giving them a rough appearance. Acting on instructions received through messenger RNA, the ribosomes engage in the synthesis of proteins. Their behaviour during the reactions of the cell following injury to the axon is of great interest (page 89).

In addition to the Nissl bodies, the cytoplasm of the motoneurone contains many mitochondria (page 6). There are also neurotubules and neurofilaments, most of which enter the dendrites and the axon. The Golgi apparatus is the name given to a system of flattened tubular channels to be found near the nucleus; one of its functions is thought to be the ‘packaging’ of proteins prior to their delivery into the axon (page 12). Finally, with advancing age, the motoneurone cytoplasm contains increasingly large masses of pigment, the ‘lipofuscin’ granules; the significance of this material is unknown.

Dendrites

The motoneurone possess several dendrites which radiate from the cell body in dorsal, superior and inferior directions; the dendrites become progressively narrower and also divide into several branches. As already stated, the function of the dendrites is to receive information from other nerve cells. They do so by establishing synaptic connections with the terminal twigs of the axons from these cells. The fine structure of these synapses is similar to that of the neuromuscular junction (Chapter 4); the terminations of the axon twigs are expanded to form ‘boutons’ inside which many synaptic vesicles can be distinguished. The packing density of the boutons on the dendritic membrane increases as the dendrites narrow; more proximally there are gaps into which processes from the glial cells project. Some synapses are also found on the membrane of the soma.

Glial cells

The glial cells of the central nervous system do not conduct impulses and therefore have no direct role in signalling. Their importance lies in their various supportive functions; they provide a structural matrix for the neurones and also control the passage of substances from the capillaries into the neuronal milieu. In addition it appears that the metabolism of the glial cells is at least partly linked to that of the neurones. In the case of the motoneurone, several oligodendroglial cells can be seen clustered around its periphery and occupying any spaces available between the synaptic knobs (boutons) of the incoming fibres.

Axon

The axon arises from a conical protrusion of the motoneurone soma known as the axon hillock and extends to the muscle as a long axis cylinder. The cylinder is bounded by a membrane, the axolemma, which has structural and functional features similar to those of the muscle fibre sarcolemma (page 3). Within the cytoplasm of the axis cylinder is an array of small neurotubules and neurofilaments thick. An interlacing system of rather wider tubules forms the endoplasmic reticulum. All the motor axons have lipid coverings surrounding the axis cylinders; these are the myelin sheaths and they are derived from a type of satellite cell known as the Schwann cell. The motor axons in man are not quite as thick as those in the cat and monkey; if the myelin sheaths are included in the measurements, their diameters range from approximately 2 to 14 μm. In the ventral roots, which contain motor fibres only, the axon diameters fall into a bimodal distribution with a separation at about 7 μm. The population of fibres with large (7–14 μm) diameters corresponds to the alpha-motor axons supplying the extrafusal muscle fibres; the smaller axons are the gamma or ‘fusimotor’ axons innervating the intrafusal muscle fibres inside the muscle spindles.

The myelin sheath has been studied with the electronmicroscope and has been shown to consist of spiral wrappings of tightly-packed membranes laid down by the Schwann cell (Geren, 1954). The myelin sheath is not a continuous structure but is interrupted at regular intervals by the nodes of Ranvier. The distance between two successive nodes is an internode and corresponds to the territory of a single Schwann cell. At birth the internodes measure about 230 μm in human motor nerves; as the limbs grow so the lengths of the internodes increase, since the number of Schwann cells remains the same. Vizoso (1950) found that the longest internodes in the ulnar and anterior tibial nerves of an 18-year-old subject were 1100 and 980 μm respectively, indicating that there had been a fourfold increase in their lengths. If, during the course of a disease process, the myelin sheath is destroyed and then remade, or if the axon is damaged and then regenerates, many of the newly formed internodes are much shorter than in the normal adult (Figure 2.2). The explanation for this discrepancy is that during the recovery process the Schwann cells divide and thereby increase their number; each new cell occupies a smaller length of axon.

Figure 2.2 Measurements of internodal length (distance separating two successive nodes of Ranvier) made on teased fibres of anterior tibial nerves from an 18-year-old girl (upper) and an 80-year-old man (lower). Notice the linear relationship between fibre diameter and internodal length in the younger subject and the deviation from this relationship (− − − −) in the older one. (From Vizoso (1950) by courtesy of the author, and the Editor and publishers of Journal of Anatomy)

The electronmicroscope has also been invaluable in revealing the ultrastructure of the nodes of the Ranvier. Figure 2.3 summarizes, in diagram form, the findings of Williams and Landon (1967). On the left-hand side of the figure the axon has been bisected by a vertical incision and a segment removed; to the right of the node some of the basement membrane has been peeled away. It can be seen that the myelin sheath on the other side of the node (paranodal region) is furrowed and that the superficial grooves are filled with columns of Schwann cell cytoplasm rich in mitochondria. As these columns approach the node they first become confluent and then send finger-like processes toward the nodal portion of the axolemma. The processes are embedded in an amorphous extracellular material known simply as ‘gap substance’. Studies by Landon and Langley (1971), among others, have demonstrated that the gap substance is composed of mucopolysaccharides, and that the anionic groups of these molecules exert a powerful electrostatic attraction for cations. Landon and Langley further suggest that the gap substance may serve to maintain a high concentration of sodium ions available for flow across the axolemma during the action potential. Similarly the gap substance might limit the diffusion of potassium from the vicinity of the node following an impulse and hold it in readiness for active transportation back into the fibre subsequently (page 21). It is also tempting to assign a functional role to the Schwann cell processes which project to the nodal axolemma. It is possible that these convey ATP to the nerve membrane from the mitochondria in the columns of Schwann cell cytoplasm; the ATP might then be used to supply the energy required for sodium ion pumping (see Landon and Langley, 1971). Another interesting feature of the myelin sheath are the Schmidt–Lanterman incisures (clefts). At each of these regions a narrow cytoplasmic process is sent from the Schwann cell to the axis cylinder. The projection does not actually penetrate the myelin wrappings but instead winds its way around the axon, following the plane of separation between the myelin lamellae. Using time-lapse cinematography Gitlin and Singer (1974) have observed that the Schmidt–Lanterman clefts are not static structures but can be open or closed at different times. It is possible that the open phase enables materials to be passed between the axis cylinder, on the one hand, and the Schwann cell and the endoneurial space of the nerve fibre, on the other. Even the remainder of the myelin sheath is not stationary but can be seen to develop small indentations which then regress; possibly this type of movement is related to some component of axoplasmic flow (see below and Weiss, 1969).

Figure 2.3 Structure of a node of Ranvier and the adjacent regions of nerve fibre (paranodes). See text for description. (From Williams and Landon (1967), courtesy of the authors, and the Editor and publishers of Gray’s Anatomy)

In considering the structure of a peripheral nerve, mention must also be made of the connective tissue. Each of the nerve fibres is enclosed in a sheath of connective tissue, or endoneurium. Within the nerve trunk the fibres are collected into a number of bundles or fasciculi, each being bounded by a condensation of connective tissue termed the perineurium. Lastly, there is a relatively thick coat of connective tissue which invests the whole nerve trunk; this is the epineurium.

AXOPLASMIC FLOW

The function of the axon is to act as a communication pathway between the spinal cord and muscle. Rapid signalling to the periphery is required if the muscle is to contract and is achieved by the nerve impulses; the ionic mechanisms underlying this event are considered in detail on page 21. Equally important, however, is a slow signalling system which enables messages, coded in the form of chemicals, to be sent in both directions between the cell body of the motoneurone and the muscle fibres which it innervates. These messages are of paramount importance for the maintenance of the normal structure and cellular metabolism of both the muscle fibre and the motoneurone; the integrity of the Schwann cells and of the axon itself are also dependent on this chemical signalling system. These controlling effects are described as ‘trophic’ and they are analysed in Chapters 8, 9 and 10. Of immediate concern is the nature of the axoplasmic transport which makes this chemical signalling system possible. It will be shown that in the centrifugal direction (from motoneurone to muscle) there are both fast and slow transport systems while in the reverse (centripetal) direction the axoplasmic flow has an intermediate velocity.

Centrifugal transport

‘Slow’ axoplasmic flow

The earliest intimation that there was normally a flow of cytoplasm from the cell body of the motoneurone outwards along the axon came from a series of experiments by Weiss and his colleagues in the 1940s (Weiss, 1944; Weiss and Davis, 1943; Weiss and Hiscoe, 1948); the results of these and later experiments have recently been summarized by Weiss (1969). The technique employed by these workers was to apply a gentle constriction to the sciatic nerve of an animal by investing the nerve with a sleeve of artery. As the arterial cuff gradually contracted, due to its own elasticity, the nerve fibres underwent compression to varying extents and the chronic effects of this were studied after several months. It was found that the segment of nerve abutting the proximal end of the constriction was swollen, partly because of oedematous fluid between the nerve fibres but mainly on account of distortions and distensions of the axons themselves. With the light microscope the axis cylinders appeared to be either beaded or else ballooned, telescoped or ‘corkscrewed’. Within and beyond the constriction the axis cylinders were narrowed. To Weiss and his colleagues the appearances of the axons suggested that there had been a ‘damming up’ of axoplasm at the site of constriction; this conclusion implied that, under normal circumstances, axoplasm must be manufactured in the cell body and somehow propelled down the axon. Supporting evidence for Weiss’s interpretation came from a subsidiary experiment in which the constriction was removed (Weiss and Cavanaugh, 1959). The dammed-up material was then released and could be observed to travel down the axon as a wave with a velocity between one and several millimetres per day. Even during the period of the constriction, however, there had been a small flow of axoplasm distally from the core of the swollen proximal region of nerve. To Weiss these results indicated that the axons were perpetually growing from the cell bodies of the motoneurones and that the contents of the new axoplasm were then consumed distally as part of a continual replenishment process. The relatively large amount of material requiring synthesis would account for the high turnover of RNA in the cell bodies of the motoneurones (and other neurones).

Although some of the conclusions reached by Weiss and his colleagues have been challenged recently by Spencer (1972), on the basis of electronmicrographs of ligated nerve, the existence of ‘slow’ axoplasmic flow is not seriously doubted. The study by Weiss and Cavanaugh (1959) has already been cited and further supporting evidence has come from the results of isotope experiments. For example, Droz and Leblond (1963) showed unequivocally that protein labelled with [³H]-leucine moved distally in rabbit sciatic nerve with a velocity of about 1 mm per day.

‘Fast’ axoplasmic flow

After the clear demonstration of axoplasmic flow with the ‘damming’ experiments described above, attempts were made to explore the phenomenon more fully by labelling the axoplasm with radioactive isotopes and then studying their passage down the axon. The first experiments involved radioactive phosphorus while later ones employed labelled carbon in glucose and amino acids; these methods have been superseded following the availability of tritiated amino acids. At present a commonly used strategy is to injected [³H]-leucine into the ventral grey matter of the lumbar region of the spinal cord; the amino acid is then taken up by the cell bodies of neighbouring motoneurones and rapidly incorporated into newly synthesized proteins. At a given time after injection the sciatic nerve is excised and cut into segments of uniform length. The amount of radioactivity in each segment is then measured with a scintillation counter and expressed as a function of distance from the spinal cord. An example from the work of Ochs and Ranish (1969) is given in Figure 2.4, the lower curve of which shows the distribution of radioactivity in the sciatic nerve of the cat 6 hr after injection of [³H]-leucine into the L7 segment of the cord. Included for comparison, and displayed in the upper curve, are the results for sensory nerve fibres following an injection given at approximately the same time into the L7 dorsal root ganglion on the opposite side. The peak of radioactivity in the sensory fibre curve corresponds to isotope remaining in the ganglion; similarly the high value at the left of the motor fibre curve denotes the activity left in, and around, the cell bodies of the motoneurones. The arrows at the bottoms of the two curves indicate the advancing fronts of the labelled axoplasm; the spatial disparity corresponds to the extra length of the motor pathway due to the inclusion of the L7 ventral root. Studies such as these have demonstrated that, in addition to the slow movement of axoplasm already described, there is a very much faster flow with a maximum velocity, in mammals, of about 410 mm/day. Another technique is to produce a temporary arrest of axoplasmic flow by cooling a short length of nerve; the transported material then accumulates in, and proximal to, the cooled region. Upon rewarming the nerve, the movement of the material is resumed and its velocity can be determined. This ‘stop-flow’ method has been used by Brimijoin (1975) to study the passage of the enzyme dopamine-β-hydroxylase in rabbit sciatic nerve; a flow rate of 300 ± 17 mm/day was found. The presence of a ‘fast’ axoplasmic flow is a property of all the motor and sensory axons which have been tested to date. Although the velocity of the ‘fast’ axoplasmic flow is independent of fibre diameter it now appears that it can be influenced quite markedly by certain drugs; for example pargyline, a monoamine oxidase inhibitor, can increase the rate fivefold (Boegman, Wood and Pinaud, 1975). Such a finding raises the obvious possibility that the flow can be modulated under physiological circumstances, but this remains to be proven (see, however, page 86). Finally, it should be noted that the neurone soma sends cytoplasm not only into the axon but also out into the various dendrites and their branches.

Figure 2.4 ) of cat sciatic nerve, measured 6 hr after injection of labelled material into dorsal root ganglion and ventral horn. See text for description of technique and results. (Modified from Ochs and Ranish (1969) courtesy of the authors, and the Editor and publishers of Journal of Neurobiology)

Mechanism of fast axoplasmic flow

By injecting inhibitors of protein synthesis such as puromycin or cycloheximide it has been shown that the motoneurone soma is able to complete its synthesis of protein within 10–20 minutes of the arrival of the labelled precursor, [³H]-leucine. Not all the amino acid is converted into protein since polypeptides are also labelled and there is a large fraction which is taken up by small axoplasmic particles.

Other studies have shown that lipids and polysaccharides are also rapidly conveyed down the axon. Included among the transported proteins are the enzymes acetylcholinesterase and choline acetyltransferase (page 31); in addition Miledi and Slater (1970) have shown that a fast-travelling factor(s) is necessary for the maintenance of the axon terminal at the neuromuscular junction. Other substances, as yet unidentified, supervise the metabolic machinery of the axon, Schwann cells and muscle fibres in keeping with the ‘trophic’ role of the motoneurone (see page 72). Of the particulate matter transported down the axon, the most prominent fraction is the mitochondria and these can be observed in motion with the phase-contrast and interference microscopes. The suggestion has been made that the mitochondria are transported to the axon terminal for the synthesis of acetylcholine, and that their ageing components are continually resynthesized from proteins conveyed by fast axoplasmic transport.

A further proposal is that the Golgi apparatus of the cell body acts as a ‘gate’ by controlling the delivery of the material into the axon (Ochs, 1972). So far as the transport mechanism itself is concerned, a number of observations are relevant. First, axoplasmic flow is able to proceed in both directions at a normal rate even when the axon has been removed from the body; this finding indicates that the transport system obtains its energy from local sources in the axon (or the Schwann cell). Secondly, on the basis of experiments utilizing metabolic blocking agents such as DNP (dinitrophenol) it is likely that ATP (backed up by creatine phosphate) provides this energy.

There is general acceptance of the idea that the neurotubules and neurofilaments within the axon are somehow involved with fast axoplasmic transport but there is no agreement as to mechanism by which this is effected. One proposal, that by Ochs (1972, Ochs 1974), is that the materials are conveyed on ‘transport filaments’, rather like wagons on a railway track. In this case the ‘track’ would be a neurotubule or neurofilament from which projecting cross bridges, energized by ATP, would move the ‘wagon’ onwards. The concept is thus very similar to the sliding filament mechanism of muscle contraction (page 35) and it is relevant to the hypothesis that actomyosin has been shown to be component of brain tissue (Berl and Puszkin, 1970). It is also probable that the system of fine interlacing longitudinal channels within the axis cylinder, the endoplasmic reticulum, is important in transporting material along the axon (Droz et al., 1975).

So far no consideration has been given to the axoplasmic flow which occurs in a centripetal direction, that is, from the muscle fibre to the motoneurone. Evidence for such a movement has come from several sources. For example, Lubinska and Niemierko (1970) have found that acetylcholinesterase is transported proximally as well as distally; the rate of the centripetal flow is about one half of the centrifugal one. Other workers have observed particles, probably mitochondria, being carried in axons away from the periphery. Proof that some of the material reaches the cell bodies of the motoneurones has been obtained by Glatt and Honegger (1973) who injected albumin coupled with the fluorescent dye, Evans blue, into triceps muscles of the rat forelimb and were able to detect dye in the ventral horns some 12 hr later. In the study by Kristensson and Olsson (1971) horseradish peroxidase was used as a marker instead. This substance had the advantage that it could produce an electron-dense reaction product and, with the electronmicroscope, its intracellular location was seen to be in small cytoplasmic granules around the nucleus. Just as the centrifugal axoplasmic flow largely mediates the trophic control of axon, Schwann cell and muscle fibre by the motoneurone, so the centripetal flow exerts a trophic influence on the motoneurone soma by the periphery. Interruption of this trophic influence, as in cutting the axon, sets in train the phenomenon of chromatolysis in the motoneurone (see page 89).

Chapter 3

RESTING AND ACTION POTENTIALS

Publisher Summary

This chapter provides an overview of the resting membrance potential and action potentials. In the case of inactive mammalian muscle, the inside of the cell has been found to be some 85 mV negative with respect to the outside; this potential difference is termed the resting membrane potential. The chapter highlights that the ability of the membranes to develop transient changes in potential, which can be transmitted from one point to another, confers upon nerve and muscle fibers’ special property of excitability. The propagated change of membrane potential is the action potential, or impulse. To record its full amplitude, it is necessary to measure, during excitation, the potential developed between an electrode in the cytoplasm of the fiber, and an external electrode. Like the resting membrane potential, the action potential has an ionic basis, depending on the permeability of the membrane to the ions on its inner and outer surfaces, as well as upon the concentrations of those ions.

It has been known for more than a century that a difference in electrical potential normally exists between the inside of a cell and its fluid environment. During the past three decades it has been possible to determine this potential difference directly by inserting a microelectrode through the cell membrane into the interior of the fibre and connecting the other terminal of the voltage measuring device to a reference electrode in the fluid bathing the cell (see Appendix 3.1 for methods). In the case of inactive mammalian muscle the inside of the cell has been found to be some 85 mV negative with respect to the outside; this potential difference is termed the resting membrane potential (Figure 3.1, left).

Figure 3.1 (Left) Resting membrane potentials recorded in situ from the brachial biceps and quadriceps muscles of three healthy human subjects (upper) and from the gracilis muscles of normal mice (lower). The greater scatter of the human results probably resulted from unavoidable damage to some of the fibres during removal of the superficial connective tissue. Data of McComas et al. (1968b). (Right) Membrane potential in three different specimens of human intercostal muscle, studied in vitro. The effect of varying the external concentration of potassium is shown. The straight line is a plot of the potassium equilibrium potential for [K]i = 156 mM at 37 °C. Each point represents the mean value from five fibres. The small deviation between the observed and predicted values, when the [K]o is less than 10 mM is due to sodium permeability of the membrane; the latter is effectively abolished by adding protein to the bathing solution; see text. (From Ludin (1970) courtesy of the author, and the Editor and publishers of European Neurology)

IONIC BASIS OF THE RESTING MEMBRANE POTENTIAL

An understanding of the resting membrane potential requires knowledge of the ions present on the two sides of the membrane. Inside the muscle fibre the most common cation is potassium; the anions are supplied by phosphate, sulphate and bicarbonate together with amino acids, polypeptides and proteins. Outside the cell the interstitial fluid has a composition similar to plasma; sodium and choloride are the dominant cation and anion respectively and smaller amounts of potassium, calcium, magnesium, bicarbonate and phosphate are also present.

The concentrations of the various ions in mammalian skeletal muscle and in its extracellular fluid are given in Table 3.1. The intracellular values should be recognized as very approximate ones for there is difficulty in determining the proportion of muscle fluid which is intracellular rather than extracellular. Further, even if the intracellular value was accurately established it would only reflect a gross average for the various organelles, myofibrils, tubular systems and cytoplasm of the fibre.

TABLE 3.1

Ionic Compositions of Mammalian Skeletal Muscle Fibres and of the Interstitial Fluid Surrounding Them

There are two reasons why the compositions of the intracellular and extracellular fluids are so different. First, a large proportion of the organic anions in the interior of the fibre are part of the cell structure and are therefore unable to migrate. Second, the resting membrane is only semi-permeable; it allows potassium and chloride ions to diffuse through but it impedes the passage of sodium. The internal anions exert an electrostatic attraction for cations which can only be satisfied by a high internal concentration of potassium, since sodium ions are unable to cross the resting membrane. The external sodium ions will, however, be effective in balancing the anions of the extracellular fluid, the most prevalent of which is chloride. This unequal distribution of potassium and chloride ions is termed a Donnan equilibrium, such