William L. Nastuk

Immunofluorescence Demonstration of a Muscle Binding, Complement-Fixing Serum Globulin Fraction in Myasthenia Gravis.

Summary 1. A muscle binding, complement fixing component has been demonstrated in the crude globulin fraction of a pool of serum from 10 patients with myasthenia gravis of recent onset and progressive character, by means of the immunofluorescence technic. This component could not be demonstrated in a similarly prepared normal serum globulin pool. Untagged myasthenic globulin was shown to block competitively adherence of fluorescein tagged myasthenic globulin to skeletal muscle striations; whereas prior treatment of muscle sections with normal serum globulin intensified staining with fluorescein tagged myasthenic globulin. Individual myasthenia gravis serums included in the pool also blocked staining with fluorescein conjugated myasthenic globulin. Normal serums did not block adherence of the fluorescein tagged myasthenic globulin to skeletal muscle. 2. The myasthenia gravis globulin fraction was shown to fix guinea pig complement to human skeletal muscle, by successive treatment of muscle sections with myasthenic globulin, guinea pig complement, and fluorescein conjugated rabbit anti-guinea pig complement. Normal serum globulin failed to fix complement by this technic. 3. Thirty-one myasthenia gravis serums were screened, with 11 normal serums, and 5 serums from patients with other generalized myopathies, for their ability to fix guinea pig complement to skeletal muscle. Thirteen myasthenic serums gave unequivocal evidences of complement fixation, using the immunofluorescence technic. No normal serums fixed complement. Serum from one patient with paroxysmal myoglobinuria also fixed complement. Serum from one patient with acute dermatomyositis gave rise to fluorescence in the sarcolemma when layered onto skeletal muscle followed successively by guinea pig complement and fluorescein conjugated rabbit anti-guinea pig complement.

An Instrument for the Production of Microelectrodes Used in Electrophysiological Studies

The membrane potential of single muscle fibers may be measured using an internal microelectrode of tip diameter less than 0.5μ. Previously such microelectrodes could only be prepared manually and this method has certain disadvantages. The problem has been overcome through the use of an instrument for which details of design, construction, and operation are given. In principle, a 35‐mm length of Pyrex tubing of outside diameter 2 mm and inside diameter 1.3 mm is heated from a surrounding electrically energized platinum coil. At the start, 100‐g force is applied to the tubing in the longitudinal direction by means of a solenoid and plunger arrangement. As the tubing softens its length is extended by 5 mm whereupon the automatic application of 1700‐g force draws the tubing apart thus forming two micropipettes. The dimensions and electrolytic resistance of typical microelectrodes manufactured with the aid of the instrument are presented.

Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction.

1. Reversible depletion of synaptic vesicles from frog cutaneous pectoris neuromuscular junctions was studied by application of a Ringer solution containing 115 m M‐K propionate.

Some ionic factors that influence the action of acetylcholine at the muscle end-plate membrane.

The essence of a widely held view of neuromuscular transmission is as follows: (1) the arrival of a motor nerve action potential greatly enhances the release of acetylcholine from the motor axon terminals; (2) this chemical intermediary diffuses to the outer surface of the end-plate membrane where it combines with receptors located there; and (3) as a consequence, the end-plate membrane develops an increased ionic permeability resulting, in this case, in a diminution of the potential difference ordinarily appearing across it. The reduction in potential difference (end-plate potential) requires the removal of a proportionate amount of the electric charge stored in the end-plate membrane capacitance. This discharge, which involves displacement of ions, can be produced by a net influx of positive charge, a net efflux of negative charge, or both. When one attempts to explain the basic mechanism involved in this transmembrane ionic displacement, two questions arise: (1) What is the identity of the moving ions? (2) At what rates do the various ionic species move across the activated end-plate membrane and how do these rates vary with time? A few years ago, Fatt and Katz (1951) proposed that the application of acetylcholine transforms the end-plate membrane into a structure that is permeable to all ions. This special patch of activated membrane is electrically coupled to the surrounding nonend-plate membrane; hence it will act as a short circuit for the latter, thereby leading to the generation of propagated muscle action potentials. In this theory the difference in potential across the activated end-plate membrane will approach the value representing the liquid junction potential between intracellular and extracellular fluids. More recently, del Castillo and Katz (1956) have reviewed the lines of evidence supporting the “short circuit” hypothesis. Sodium ions represent a likely source of rapidly penetrating positive charge required to generate the end-plate potential because their chemical gradient and the electric field in the inactive membrane favor inward movement. That additional ion species are involved was indicated by the early work of Fatt (1950) in which it was shown that in the absence of extracellular sodium ions, the acetylcholine-activated membrane becomes depolarized, although to a more limited extent. With the idea of removing certain difficulties inherent in the work of Fatt (19jO), I repeated his experiments in sodium-free Ringer’s solution, but used intracellular recording and a high-speed microelectrophoretic method for apply-

Development of a High‐Fidelity Preamplifier for Use in the Recording of Bioelectric Potentials with Intracellular Electrodes

In the intracellular recording of action potentials, the fidelity of recording is limited by the resistance and capacitance of the ultramicroelectrode required for making connection to the intracellular contents, and by the characteristics of the input circuit of the amplifier. The cathode follower circuit conventionally used to minimize the time constant of the recording system is discussed and its shortcomings are analyzed. An improved circuit involving positive feedback is presented. The measurement of the recording system time constant is discussed.

Muscle Postjunctional Membrane: Changes in Chemosensitivity Produced by Calcium

Increases in the extracellular concentration of calcium ions above 1.8 millimoles per liter caused a reversible decrease in the sensitivity of tmuscle postiunctional membrane to carbamylcholine. A quantitative study of the inhibitory effect of calcium ions on membrane depolarization produced by carbamylcholine indicates that calcium ions compete with carbamylcholine for some common binding sites on the postjunctional membrane. Calcium ions (20 millimoles per liter) caused a neuromuscular block wherein prolonged end plate potentials were produced after nerve stimulation. Calcium ions applied ionophoretically to the postjunctional membrane decreased the amplitude and prolonged the time course of the transient depolarization produced by ionophoretically applied carbamylcholine.

THE MECHANISMS UNDERLYING NEUROMUSCULAR BLOCK FOLLOWING PROLONGED EXPOSURE TO DEPOLARIZING AGENTS

Our knowledge of neuromuscular physiology has been greatly expanded in recent years by study of drug action a t the myoneural junction. One type of neuromuscular block that has been much studied is the one which follows prolonged application of the quaternary ammonium compounds. This block is particularly interesting because its intensity and characteristics vary during the sustained drug application. In the study of transmission blocks of this kind, a widely used method is to record the isometric tension produced by an isolated nerve-muscIe preparation when single stimuli are applied to the motor nerve. The most frequently depicted result of such studies is shown diagrammatically in FlGUKE 1. Following the application of the depolarizing drug, there is a rapid decrease in tension output. Following this, the tension output increases, bu t with time it slowly declines once again. Various descriptive terms have been applied to such a diphasic curve of tension output. Jenden (1951, 1954) labeled the early and late periods of decreased tension output “Phase I” and “Phase 11,” respectively. He reported that Phase I was little affected by alteration of the potassium ion concentrat,ion of the bathing fluid, by variat,ions in temperature or by addition of cholinesterase inhibitors. Phase I1 was strongly influenced by these experimental maneuvers. LJsing various laboratory species, Zaimis (1953) investigated the effect, on tension output , of prolonged applicat,ion of decamethoniiini (C,”) to neurally stimulated muscle in uiuo. She labeled the neuromuscular block produced, “Dual Block,” hecause with time the block appeared to change its properties. A t first it resembled that produced by depolarizing drugs, but later it appeared more like that, produced by “competitive” drugs such as d-tubocurarine. On the basis of such work, many workers have customarily labeled Phase I a depolarizing block and Phase I1 a competitive block (or curare-like), and it is common practice to use this relatively simple classification, although Creese (1963) has pointed out t,he many ways in which Phase I1 block is not curare-like. Our basic understanding of Phase I1 neuromuscular block will not be complete until

Sarcomeric oscillations in frog skeletal muscle fibers.

Brief asynchronous, small-amplitude, cyclic, longitudinal displacements of the striations of frog skeletal muscle fibers were observed with ordinary light microscopy after application of caffeine and certain quaternary ammonium compounds. With time these oscillations became synchronized and evolved into peristaltic-like movements. The oscillations were influenced by sarcomere length, temperature, external concentration of calcium ions, membrane potential, and disruption of the transverse tubules.

FUNDAMENTAL ASPECTS OF NEUROMUSCULAR TRANSMISSION

w.e may begin by reviewing the steps in neuromuscular transmission as shown in Fig. 1. It is now widely accepted that acetylcholine (ACh) is the transmitter at the neuromuscular junction. Hebb has recently reviewed the evidence that ACh is synthesized and stored in neuronal terminals. She has detailed the arguments to support the view that choline acetylase is located in synaptic vesicles, and that these vesicles are storage depots for ACh. There are many technical problems in the study of ACh synthesis and storage which I cannot discuss here, and an additional major difficulty in the study of the neuromuscular junction is that the important neural structures cannot be isolated and subjected to direct analysis. However, the superior cervical ganglion offers good opportunity for this kind of investigation, and it has been used by Birks and Macintosh and Birks for this purpose. Their carefully conducted experiments and thoughtful analysis of the results should be consulted by those interested in the synthesis, storage, and release of ACh from presynaptic neurons. Birks

Effect of Age and Sex on Sensitivity to D-Tubocurarine in the Rat.∗

Summary One- and three-month-old male and female rats were injected intraperito-neally with d-tubocurarine in various doses. These animals were then placed on their sides and their ability to regain the upright position determined. No difference in sensitivity to d-tubocurarine was found to exist between one-month-old females, one-month-old males, and 3-month-old males. The 3-month-old females, however, were significantly more sensitive than any of the other groups. Gonadec-tomy performed at age one week failed to abolish the sex difference in curare sensitivity in animals tested at 3 months of age. We would like to express our appreciation to Dr. A. Varma for statistical analysis of the data, and to Drs. H. Houston Merritt, and L. P. Rowland for review of the manuscript. We would also like to thank Mrs. Arleene Ozerkis, Mr. Robert Rodvien, Mr. Robert Lattes, and Mrs. Barbara Wolf for technical help. We are grateful to E. R. Squibb for supplies of Intocostrin.