Pharmacological and physiological properties of denervated mammalian skeletal muscle.

In the current work the relation between the newly formed, cholinergic membrane receptors and the Ach-induced contracture, the relation between fibrillation and denervation contracture, and the nature of the cholinergic and adrenergic receptors at end-plate-free sites were investigated in the denervated anterior gracilis muscle of the rat. The anterior gracilis muscle of the rat was selected as the test object because it is anatomically suited for the study of the pharmacological characteristics of the newly-formed receptors of endplate-free regions of the denervated muscle. A new microdrop technic was developed for the topical application of 0.5 uml volumes of drug solution to the membrane. Ach sensitivity after denervation develops at a uniform rate over the entire surface of the denervated anterior gracilis muscle. Depolarization is quantitatively related to the concentration of topically applied Ach, and the degree of depolarization is proportional to the magnitude of contracture. Denervation contracture of Ach-stimulated muscle is not the consequence of a massive discharge of a massive discharge of fibrillatory potentials, but is a non-propagated response produced by a persistent, local depolarization. The anterior gracilis muscle is pharmacologically unique because the junctional receptors of the normal muscle and the newly-formed receptors on the end-plate-free regions of the muscle are muscarinic as well as nicotinic in character. Thus, the Ach-produced contracture of the anterior gracilis muscle denervation is the result of the development of both muscarinic and nicotinic receptors over the entire surface of the muscle. In contract to the effects of Ach on the denervated skeletal muscle, catecholamines applied topically do not produce either depolarization or contracture. The only adrenergic response elicited by the topical application of catecholamines is an increase in the rate of discharge of fibrillatory potentials. However, the catecholamines administered intra-arterially produce both depolarization and contracture. Unlike Ach-induced contracture, it does not appear that catecholamine-induced contracture of denervated muscle is the result of the development of new adrenergic receptors on the membrane.

PHARMACOLOGICAL AND PHYSIOLOGICAL PROPERTIES OF DENERVATED MAMMALIAN SKELETAL MUSCLE by Stuart All en Turkan is A thesis submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology Un ivers ity of Utah June 1967 This Thesis for the Doctor of Phi losophy Degree by Stuart All en Turkan is has been approved May 1967 Chairman, Supervisory Committee Reader, Supervisory Committee Head, Major Department UNIVERSITY Of UTAti UBKAH1ES ACKNOWLEDGMENTS The author greatly appreciates the instruction that he received from all the members of the Departments of Pharmaco logy and Phys io logy. Gra'titude is par1"icu larly expressed to Dr. Ralph Karler for h is warm personal interest, encouragement, and instruction not only during the thesis research but throughout my graduate training. His expertise and advice during the preparation of this manuscript deserve a special note of thanks. Sincere appreciation is extended to Dr. Carlos Eyzaguirre for recommending the topic of this thesis, for his suggestions during the thesis research, and for his i nte res tin th e au thor's p rog ress and career. The author is also grateful to Dr. Louis S. Goodman, Dr. Don W. Esplin, Dr. Carlos Eyzaguirre, Dr. Dixon M. Woodbury, and Dr. Richard Orkand for their suggestions in the preparation of the final manuscript. The author is indebted to Dr. Arthur Hess for preparing the anterior graci I is muscles stained for cholinesterase. Appreciation is extended to Mr. Max Steadman, Mr. Thomas 0 'Leary, and Mr. Aldo Gabardi for their help in the construction and maintenance of the equ ipment used in the investigation. Thanks are also due to Mr. Pierre Leno ir for mak ing the photograph ic repro­ductions of the experimental responses shown in this thesis and for his excellent care of the experimental animals. iii I am particularly grateful to Mrs. Marya Bejar for her expeditious and careful typing of the numerous rough drafts of this thesis and to Lou Ann Robinson for expert typing of the final copy. My wife, Carolyn, deserves special thanks for her understanding and en­couragement throughout my many months of research and writing. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ABSTRACT I. INTRODUCTION A. Historical Background Page iii vii B. Statement of the Probl em 5 II. METHODS 10 A. Prepara1"ion 10 1. Anatomy and innervation 10 2. Histo log ica I exam ination 10 3. Dissec tion of the preparation 11 4. Denervation 11 B. Recording Technics 12 1. Muscle tension 12 2. Capac itance-coup I ed record ing of action potentia Is 12 3. DC recording of potentials 13 4. Temperature monitoring and recording 14 C. Adm in istration of Drugs 14 D. Criteria for Experimental Procedures 17 E. Experimental Conditions for Neural Stimulation of the Muscle 18 III. RESULTS 20 A. Fibrillatory Activity of the Denervated Anterior Gracilis Muscle 20 B. Development of ACh-induced Contracture 20 C. Effect of ACh on Extracellularly Recorded Fibri lIatory Potentials 22 v Page D. Relationsh ip Between Depolarization and the Concentration of Topically Applied ACh 23 E. ACh-induced Depolarization and Contracture 25 F. Local Nature of ACh-induced Responses 27 G. Effects of ~-Tubocurarine (DTC) 27 H. Effects of Atropine 28 I. Effects of Norepinephrine and Isoproterenol 29 IV. DISCUSSION 31 V. SUMMARY 49 VI. REFERENCES 51 VII. FIGURES 57 VIII. VITA 83 IX. RESEARCH PROPOSALS 84 A. Effects of Atropine on Neuromuscular Transmission in the Anterior Grac iI is Musc Ie of the Rat 84 B. Denervation Contracture Produced by Catecholamines 85 C. The Troph ic Influence of Motor Nerves on Skeletal Muscle 86 D. Research Proposa I References 88 vi ABSTRACT In the current work the relation between the newly-formed, cholinerg ic membrane receptors and the ACh-induced contracture, the relation between fibri Ilation and denervation contracture, and the nature of the chol inerg ic and adrenerg ic receptors at end-plate-free sites were investigated in the de­nervated anterior gracilis muscle of the rat. The anterior gracilis muscle of the rat was selected as the test object because it is anatomically suited for the study of the pharmacological characteristics of the newly-formed receptors of end­plate- free regions of the denervated muscle. A new microdrop technic was developed for the topical application of 0.5 to 1 fl volumes of drug solution to the membrane. ACh sensitivity after denervation develops at a uniform rate over the entire surface of the denervated anterior graci I is muse Ie. Depolarization is quantitatively related to the concentration of topically applied ACh, and the degree of depolariza­tion is proportional to the magnitude of contracture. Denervation contracture of ACh-stimulated muscle is not the consequence of a massive discharge of fibril­latory potentials, but is a non-propagated response produced by a persistent, local depolarization. The anterior gracilis muscle is pharmacologically unique because the junctional receptors of the normal muscle and the newly-formed receptors on the end-plate-free regions of the muscle are muscarinic as well as nicotinic in character. Thus, the ACh-produced contracture of the anterior vii gracilis muscle after denervation is the result of the development of both mus­carinic and nicotinic receptors over the entire surface of the muscle. In contract to the effects of ACh on the denervated skeletal muscle, cate­cholamines appl ied topically do not produce either depolarization or contracture. The only adrenergic response elicited by the topical application of catecholamines is an increase in the rate of discharge of fibrillatory potentials. However, the catecholamines administered intra-arterially produce both depolarization and con­tracture. Unlike ACh-induced contracture, it does not appear that catecholamine­induced contracture of denervated muscle is the result of the development of new adrenergic receptors on the membrane. viii I. INTRODUCTION A. Historical Background "When in a series of efferent neurones a un it is destroyed, an increased irritability to chemical agenfs develops in the isolated structure or structures, the effecfs being maximal in the part directly denervated" (Cannon, 1939). Phi I ipeaux & Vu Ipian (1863) made the first observation of denervation super-sensitivity in skeletal muscle. They described a prolonged contracture evoked in the denervated tongue muscles of the dog by stimulation of the chorda tympani or the lingual nerve. Stimulation of the chorda tympani or the lingual nerve does not el ic it a contraction in the normally innervated tongue. The phenomenon was subsequently called the pseudomotor response. Heidenhain (1883) confirmed the occurrence of the pseudomotor response in the denervated tongue muscles of the dog and noted the disappearance of the phenomenon with the reinnervation of the muscles. He also reported that the contracture is assoc iated with activation of the vasodi lator fibers in the tongue and that fibri IIation is characteristic of denervated tongue musc I es . Sherrington investigated post-denervation events in skeletal muscle and in 1894 described the results of the following experiments. He sectioned the ventral roots innervating the hind I imbs of cats and several weeks later stimu lated the degenerating sciatic nerve. Stimulation of the sciatic nerve containing intact sympathetic and dorsal root fibers produced a contracture of the denervated muscles (the Sherrington phenomenon). The work of Phil ipeaux & Vu Ipian, He idenhain, and Sherrington represenfs the major mi lestones in the description of the pseudo-motor phenomenon up to the turn of the century. 2 Lang ley (1905) made the first observation of supersensitivity of a denervated skeletal muscle to a specific chemical agent. He demonstrated that denervated fowl muscle became supersensitive to nicotine. The first explanation of the pseudomotor contracture involving increased sensitivity to a chemical agent was expressed by Lang ley in 1921. He postu lated that the pseudomotor response resu Ited from the I iberation of a substance, at the postgang I ion ic sympathetic nerve endings in skeletal muscle, that causes contracture. He also suggested that denervation sensitizes the muscle to the endogenously I iberated agent. The initial evidence for such an explanation of denervation phenomena was presented a year later in 1922 by Frank, Northmann, & Hirsch-Kaufman. They showed that stimulation of appropriate vasodilator nerves and parenteral adminis­tration of acetylcholine (ACh) produced similar contractile effects in denervated skeletal muscle. In addition, Dale & Gasser (1926) and Dale & Gaddum{l930) found that eserine enhanced denervation contracture produced either by ACh admin istered parenterally or by stimu lation of sympathetic nerves. The latter investigators suggested that the stimu lation of sympathetic vasodi lator fibers released endogenous ACh which then diffused to the denervated structure and caused a contracture. In further support of the hypothes is that ACh was involved in denervation contracture, von Euler & Gaddum (1931) demonstrated the presence of cholinergic fibers in sympathetic nerves. Hinsey & Cutting (1933) noted that the Sherrington phenomenon disappears upon section of the sympathetic nerve supply to the denervated muscle. ACh was further implicated by Bain (1932) and by Feldberg 3 (1933) who recovered ACh-like material, after stimulation of the lingual nerve, from eserinized perfusates of tongue muscles of the dog. BBlbring & Burn (1935) obtained similar results from experiments performed on the hindlimb of the dog after stimu lotion of the sympathetic innerva'Hon. The resu Its of the above studies represent the first direct evidence that denervation contracture of skeletal muscle is the result of ACh liberated from autonomic nerves. Furthermore, the work of Langley (1905) and Frank et 01. (1922) indicated the development of increased sensitivity of denervated skeletal muscle to exogenous chol inergic agents. T empora Ily parallel with the cited observations of the 1920s and 1930s were the many elegant investigations that served to establish the role of ACh as the normal neurochemical transm itter at various neuroeffector junctions in the autonomic nervous system and at the neuromuscular junctions of skeletal muscle. For example, Dale & Gasser (l926), Gasser & Dale (l926), Dale & Gaddum (l930), Dale, Feldberg, & Vogt (1936), Brown (1937), Rosenblueth & Luco (1937), and Cannon & Haimovici (1939) conducted some of their classical physiological and pharmacological studies of denervated and normal skeletal muscles by means of intra-arterially administered agents. Their work led to the fundamental conclusions that ACh is the chemical transmitter at the neuro­muscu lor junction and that denervated musc Ie is supersensitive to ACh. Some of their specific findings are presented below, in the Statement of the Problem and in the DISCUSSION. 4 Other classical observations that deserve brief consideration are the follow­ing. Bender (1938) demonstrated that the denervated facial musc les of the monkey contracted when the animal was frightened. Since the effect was enhanced by eserine, Bender conc I uded that the fright response re I eased ACh from somewhere in the body and that th is substance evoked a contracture in the denervated structures. However, von Eu ler & Gaddum had demonstrated seven years earlier, in 1931, that epinephrine can directly produce a contracture as well as enhance the pseudomotor contracture of denervated fac ia I musc les. Conse­quently, Cannon & Rosenblueth (1949) suggested that the contracture phenomenon noted by Bender may have been the result of epinephrine released from the adrenal medu Ila. Such experimental find ings demonstrate that denervated skeletal muscles become supersensitive to catecholamines as well as to the normal transm itter substance. Kuffler in 1943 studied the pharmacological properties of the chronically denervated frog sartorius by the topical appl ication of drugs. He observed that the sensitivity of the end-plate to ACh, nicotine, and caffeine increased 1000 to 100,000 times after section of the motor nerve. He also noted that end-plate­free sites in the denervated muscle were depolarized by ACh topically applied to such sites, but he attributed this response to the diffusion of ACh to end-plate areas. Kuffler cautiously concluded that the above drugs did not appreciably depolarize the end-plate-free areas of normal or denervated musc les. In contrast to the conclusions drawn by Kuffler (1943), Axelsson & Thesleff (1959) and Miledi (19600) demonstrated with iontophoretic application of ACh that the denervated end-plates of amphibian and mammalian muscle maintain their normal sensitivity to ACh. They concluded that the apparent super­sensitivity seen with diffusely applied and parenterally administered drugs is the result of an increase in the cholinergic chemosensitive area following denervation. In other words, the end-plate-free areas of denervated muscle become sensitive to ACh, whereas in normally innervated muscle such areas are insensitive to ACh. Ginetzinsky & Shamarina in 1942 demonstrated the spread of the ACh­receptor area in denervated muscles of several different species, and they also noted that fetal muscle, like denervated muscle, had an extensive cholinergic chemoreceptive area.. Unfortunately, their observations were published in Russ ian and were not brought to the attention of the Eng I ish -speak ing world until after the above-cited publications ofAxelsson & Thesleff and Miledi (see Diamond & Miledi, 1962). B. Statement of the Problem There are numerous differences that distinguish denervated from normal muscle. Some of these differences ore described in the Historical Background above and in the DISCUSSION. Great caution must be employed in developing general concepts of denervation phenomena because of the marked diversities in the experimental conditions from one study to another. Resu Its from similar tests may vary in different species as well as in different muscles even in the same spec ies. A comparison of the characteristics of the denervated frog sar­torius with those of the cat tenuissimus illustrates the problem of species 5 differences. In the former the end-plate-free regions are much less sens itive than the end-plates to ACh (Miledi, 19600); in the fatter, the end-plate-free regions approach the sensitivity of the end-plates (Axelsson & Thesleff, 1959). There are numerous experimental conditions that may also affect the results, such as the length of the distal nerve stump (Luco & Eyzaguirre, 1955; Emmelin & Maim, 1965), the route of ACh administration (Axelsson & Thesleff, 1959), and the recording techn ic 0Nare, Bennett, & Mc Intyre, 1954). The I imitations described above compl icate an attempt to formu late any general concepts of denervation phenomena, There is, however, an abundance of evidence that the phys iological and pharmacolog ical properties of the dener­vated and normal skeletal muscle membranes are different. After section of the motor nerve, there is an increase in the membrane resistance (Nicholls, 6 1956; Hubbard, 1963), an increase in the chemoreceptive area of the membrane (Axelsson & Thesleff, 1959; Miledi, 1960a; Miledi, 1962), and an increase in the activity of the relaxing factor (Brody, 1966). In addition, there is a de­crease in the accommodation of the membrane to electrical stimu lation (Desmedt, 1950), in the potassium conductance (Harris & Nicholls, 1956), and in the end­plate chol inesterase (Miledi, 1960b). Nicholls (1956) and Hubbard (1963) maintain that, despite the demonstrated differences in the electrical properties of normal and denervated muscles, none of those recognized to date can ex-plain the development of supersensitivity to ACh. The fact remains, nevertheless, that changes in the character of the membrane represent the distingu ish ing features of the denervated muscle (Gutmann, 1963). The basic problem of determ ining the relationsh ip between membrane events and the exaggerated contractile response in denervated skeletal muscle has yet to be solved. The 7 major aim of this thesis research was to study the physiological and pharmacological characteristics of" the denervated musc Ie membrane and the relation of these characteristics to denervation contracture. In normal skeletal musc Ie depolarization has been causally I inked to con­traction (Kuffler, 1946; Katz, 1950; Sten-Knudsen, 1954,1960; Huxley & Taylor, 1958; Watanabe, 1958; Hodgkin & Horowicz, 1960; Orkand, 1962), Since the properties of the denervated muscle membrane are different, a similar causal relationsh ip in denervated musc Ie cannot be assumed to exist. In fact I there is some evidence that ACh can produce a contracture in denervated skeletal muscle without producing depolarization. Jenkinson & Nicholls in 1961 re­ported that ACh produced contracture in the denervated rat diaphragm completely depolarized by potassium. Since their observations required special experimental conditions, it was decided to examine, as part of this research thesis, the relationsh ip between ACh-induced depolarization and contracture under more physiological conditions. According to the classical work of Brown (1937) and Rosenblueth & Luco (1937), a burst of musc Ie action potentia Is evoked by the intra-arterial adm in is­tration of ACh is capable of causing contraction in both denervated and normal muscle. These authors reported that denervation contracture produced by intra­arterially administered ACh was a prolonged contractile response not associated with muscle action potentials. However, Axelsson & Thesleff (1959) noted 8 that denervation phenomena, such as maximum supersensitivity, vary with the route of administration of the chol inergic agents.. In the research reported herein, the relationsh ip between contracture and action potentials was re-exam ined in the denervated gracilis of the rat by two different routes of ACh administra­tion, topical and parenteral. As previously indicated, the chemoreceptive area of the membrane spreads from the end-plate zone after section of the motor nerve (Axelsson & Thesleff, 1959; Miledi, 1960a). Axelsson & Thesleff{l959), using iontophoretically applied ACh, noted a similarity between the depolarizations of denervated muscle membrane and those of the end-plates of normal muscle.. In addi1'ion, the ACh-el ic ited depolarizations were antagon ized by ~tubocurarine (DTC). Therefore, the newly-formed receptors were assumed to be similar to those at the normal neuromuscu lar junction (Axelsson & Thesleff, 1959).. Thesleff & Elmqvist (l960) suggested that the newly-formed chemoreceptive area might serve as an excell ent model for drug stud i es . However, in most investigations the agonists and antagonists are administered parenterally or added to a bathing solution; consequently, direct pharmacological analyses of the receptors at end-plate-free sites on denervated muscle are few in number (see Ber6nek & . Vysko~iI, 1967). By use of topically applied ACh and the classical cholinergic antagonists atropine and DTC, the pharmacological nature of the receptors at end-plate-free sites on the denervated anterior graci I is was in ves 1'ig a ted in the current work. There are numerous reports that denervated skeletal muscle becomes super­sensitive to adrenergic as well as to cholinergic agents (von Euler & Gaddum, 1931; BlIIbring & Burn, 1936; luco & Sanchez, 1959; Bowman & Zaimis, 1961). It is generally accepted that the supersensitivity to chol inergic drugs is the result of an increased chemoreceptive area of the muscle membrane (Axelsson & Thesleff, 1959; Miledi, 1960a; Miledi, 1962). It is reasonable to suggest that the denervated muscle membrane may also develop adrenergic receptors over its entire surface, and such a possibility was examined in the current study. 9 II. METHODS A. Preparation The test object of the current investigation was the anterior gracil is muscle of adult, female Sprague-Dawley rats ranging in weight from 200 to 260gm. 1. Anatomy and innervation Jarcho, Eyzaguirre, Berman, & Lilienthal (1952) previously described the an­terior grac iI is musc Ie of the rat as follows. It is a long , flat, s'triated musc Ie located on the medial surface of the thigh. It originates in the inferior ramus of the pubis and inserts on the medial surface of the tibia near the knee joint. The muscle is approximately 30 mm long, 5 to 6 mm wide, and about 1 mm thick. Most of the muscle fibers extend the entire length of the muscle bundle and are usually 40 to 70JJ in diameter. The muscle is innervated by the obturator nerve, and branches of the nerve supply the two discrete zones of end-plates 1 mm wide. One end-plate zone is located near the entrance of the nerve into the muscle; the other is adjacent to and proximal to saphenous vessels (Figure 1). Each fiber contains on Iy one end-plate. 2. Histolog ical examination Two normal muscles were fixed and stained for cholinesterase by the thiocholine technic of Koelle (1951, 1955), and numerous small bundles and single muscle fibers were teased from the treated muscle and examined under the I ight microscope (Figure 1). The above-described observations of Jarcho and associates were confirmed, for example, the existence of only two end-plate zones in the muscle and their loca­tions, the presence of only one end-plate in each fiber, and the length and diameter of the fibers. 11 3. Dissection of the preparation Rats were anesthetized by intraperitoneal adm in istration of 50 mg of pento­barbital per kg and were fastened with surgical tape in the supine position to a cork board. The left leg was rotated externally and immobil ized, and the skin on the medial surface of the thigh was incised from the inguinal fold almost to the knee. Then the edges of the sk in were retracted in an upward and out­ward direction by means of hooks and elastic bands in order to form a small pool. With a minimum of dissection the gracilis muscle was exposed at the bottom of the pool. The saphenous vessels which pass perpendicularly over the muscle were tied off at the edges of the muscle. The preparatior was carefully dissected, from its insertion near the knee jo int up to a few mm distal to the main blood supply at the pelvic end of the muscle (Figure 1). The pool was filled with light mineral oil in order to prevent drying of the tissues. 4. Denervation The left obturator nerve was sectioned at the site where it disappeared under the anterior gracilis muscle, and at least 5 mm of the proximal stump of the nerve was removed in order to prevent rapid regeneration. Before each experiment the preparation was examined under the dissecting microscope to insure that the muscle was still completely denervated. During the denervation procedures the rats were anesthetized by intraperitoneal administration of 40 mg of methohexital per kg. B. Recording Technics 1. Muscle tension The distal end of the dissected muscle was fastened with a silk thread to a tension transducer which was sufficiently sensitive and stable to measure reproducibly 5 mg of tension change. The transducing device of the tension­measuring instrument is a pair of Micro-systems Type DC06A7-16-350 strain 12 gages with a gage factor of 250 (Electro-Op1"ical Systems, Pasadena, California). The transduc ing instrument was mounted on a rack and p in ion that readi Iy permitted the adjustment of the muscle to an estimate of resting length. 2. Capacitance-coupled recording of action potentials Spontaneous or fibrillatory potentials from denervated muscle and action potentials from normal muscle were led from the surface of the preparations by means of a pair of fine, platinum-iridium, wire electrodes. With a micromani­pulator, the recording leads were easily posi'l"ioned anywhere on the surface of the muscle. Electrical activity was amplified by a Tektronix Type 122, low­level preamplifier and by a Tektronix Type 3A3 amplifier. The responses were displayed on a Tektronix Type 561 oscilloscope, and photographed on moving Kodak Linagraph Ortho 35-mm film with a Grass C-4 kymograph camera. Fibrillatory potentials were continuously recorded in the above manner and monitored with an audioamplifier system in the study of contracture-threshold changes with time after denervation. In all other experiments the spontaneous potentials were mon itored intermittently. 3. DC recording of potentials Transmembrane potential differences, fibrillatory potentials, and drug­elicited depolarizations {current flow} were recorded by means of conventional 3 M KCI-filled glass microelectrodes (Caldwell & Downing, 1955) and a cathode-follower probe (Fein, 1964). An indifferent electrode that consisted of another microelectrode or a 3 M KCI-saturated cotton wick inserted in a glass capillary tube was positioned on the surface of the muscle of the opposite leg. The output of the cathode-follower probe was amplified by a Tektronix Type 3A3 amplifier and displayed on a Tektronix Type 561 oscilloscope. The responses were photographed on moving film in a manner previously described. 13 In many experiments in which slow changes in the membrane potential or slowly­rising depolarizations were recorded, the output of the probe was ampl ified by a Tektronix Type 2A63 amplifier and recorded on a Texas Instrument dual­channel polygraph. Glass microelectrodes were made with an Industrial Science Associates Model M-1 micropipette pu Iler from Pyrex number 7740 redrawn-g lass tubing with an outside diameter ranging from 1. 1 to 1 .3 mm and with a wall th ickness of 0.3 mm (~ lOOk). The glass microelectrodes were filled with 3M KCI solution as described by Caldwell & Downing (1955). Electrodes used for extracellular recording had a resistance of 3 to 10 megohms, while those utilized for intra­cellular recording were 5 to 20 megohms. With a microman ipu lator that held the electrode support, the micropipettes were easily positioned on a specific site on the membrane. By means of anatomical 14 landmarks and a reticular scale in the microscope eyepiece, the microelectrodes could be positioned repeatedly at any particular site on the muscle. 4. Temperature monitoring and recording Recta I temperature of the an imals was con1"inuous Iy mon itored throughout the experiments by means of a conventional temperature-measuring instrument consisting of a thermistor element in a probe, a Wheatstone bridge, and a temperature-cal ibrated microammeter. With a similar thermistor probe and the same bridge circuit and metering system, the temperature of the mineral oil pool was intermittently monitored during the course of the experiments. Pool temperature remained relatively constant because of its small volume of 3 to 4 ml. Rectal temperature, which normally ranged from 37 to 390 C, decreased very slowly during most experiments.. If the body temperature fell to 360 C, the heat from a 100-watt bulb in a reflector was cautiously used to maintain a rectal temperature within the normal range. Although changes in temperature of the mineral oil pool followed those in the body, the temperature of the pool WQS usually 1 .5 to 20 C lower. Room temperature was continuously recorded during the experiments by a Bacharach Industrial Instrument T empscribe and was about 260 C. C. Adm in istration of Drugs In the contracture-threshold studies, ACh solution was applied with 3-mm squares of Whatman No.5 filter paper to the surface of the denervated and normal anterior gracilis muscles. The drug-soaked filter papers were positioned with a pair of fine-pointed forceps and with observation through a dissecting micro­scope. 15 For the other experiments involving the topical application of drugs, the following unique microappl icator system was designed in order to apply un iform microdrops of d rug sol ution to the preparation. The instrument, as illustrated in Figure 2, consisted of a Hamilton PB600-1 repea1"ing dispenser (The Hamilton Company, Whittier, California), a 25 or 50 pi Hamilton gas-tight syringe, a fine bore polyethylene tubing, and a blunted 27-gauge hypodermic needle. The Hami I ton repeating dispenser ejected one-fiftieth of the total vo lume of the syringe, and the microsyringe was connected by means of the polyethylene tubing to the hypodermic needle. The needle was supported by an electrode holder attached to a micromanipulator; consequently, the needle used for the application of microdrops could be accurately placed on the membrane. The tip of the drug applicator was placed adjacent to the recording el,ectrode, and a microdrop of solution was carefully ejected in order to surround the recording electrode. This method of drug appl ication was used to el ic it depolarizations recorded extracellu larly and intracellu larly 0 The microdrop of drug solution was easily removed from the surface of the muscle by a miniature cotton pellet moistened with 0.9% NaCI solution. In experiments in which the drug solution was applied repeatedly to the same site, the area of appl ication was washed with 0.90/0 NaCI solution three times. At least 20 min was allowed between applications to the same site. 16 Some drugs were administered intra-arterially in the direction of the heart by means of a cannula inserted into the contralateral external iliac artery. The procedure was employed to avoid impairing the blood supply to the dener­vated muscle. In addition, some drugs were administered intravenously by means of the femoral vein. The following drugs were used: sodium pentobarbital (Abbott Laboratories, Chicago, Illinois), sodium methohexital (Eli Lilly and Company, Indianapolis, Indiana), acetylcholine chloride (Merck and Company, Inc., Rahway, New Jersey, and Sigma Chemical Company, St. Louis, Missouri), atropine sulfate (Sigma Chemical Company and A. H. Robins Company, Inc., Richmond, Virginia), ~-tubocurarine chloride (Eli Lilly and Company), !..-norepinephrine bitartrate monohydrate (Sterl ing-Winthrop Research Institute, Rensselaer, New York) , and !..-isoproterenol ~-bitortrate dihydrate (Sterl ing-Winthrop Research Institute). The doses and concentrations of the drugs used in the present work refer to the free-form rather than the sal t. The drug solutions were freshly prepared in 0.9% NaCI solution at the time of their use and were administered parenterally or appl ied topically at room temperature. Animals treated with DTC were artificially respired as needed with a Palmer small-animal respiratory pump. The rate of the fibrillatory potentials, which is extremely sensitive to changes in respiratory function, was utilized as a guide to determine the parameters for artificial respiration in experiments on denervated muse Ie. D. Criteria for Experimental Procedures Certain bas ic criteria for the preparations, the recording electrodes, and the appl ication of drug solution were developed in an attempt to standardize the procedures of the present investigation. The physiological condition of the preparation was a prime consideration. 17 Fibrillatory movements or potentials were monitored in all experiments on denervated muscle because fibrillation is an excellent index of the physiological status of the preparation (see DISCUSSION). If the untreated denervated muscle was not fibrillating or stopped fibrillating during an experiment as a result of deterioration, the preparation was discarded. Furthermore, during experiments in which drugs were parenterally administered, blood pressure was monitored by means of a cannula in the right carotid artery and a Tycos manometer. When the mean blood pressure was less than 70 mm Hg, the preparation was usually non-functional. Before any drugs were tested, the glass microelectrodes used for extra­cellular recording were required to maintain an extremely steady baseline while in contact with the muscle. As an additional control, a microdrop of 0.90/0 Na CI solution was positioned at the tip of the electrode. If the control solution did not evoke a response, then the electrode was util ized in the exper i men t . A small percentage of denervated muscles responded with small depolariza­tions or hyperpolarizations to the repeated application of O. 90k NaCI solution 18 and sl)ch preparations were not used in the current work. Normal musc les examined by the microdrop technic yielded inconsistent responses to the con'trol test with the 0.9% NaCI solution; consequently, normal muscles were not studied by the microdrop method. The end-plate zones were readily located anatomically and func1"ionally by following the degenerating nerve and by recording intracellularly a fibrillatory potential preceded by a prepotential. The prepotentials of the denervated rat anterior gracilis muscle are generated only at end-plate zones (Belmar & Eyzaguirre, 1966). The locations of the end-plate zones by means of functional, gross anatomical, and histological criteria agree favorably. The manner in wh ich the m icrodrops were appl ied partially determined the reproducibility of the results. It was essential to depress gently the triggering mechanism of the repeating dispenser because it was impossible to place a rapidly ejected drop accurately on the muscle. In spite of the precautions taken to position the microdrop accurately, the geometry of the muscle can cause the drop to move away from the recording electrode. Therefore, great care was taken to maintain the muscle as flat as possible to minimize movement of the drops. Responses that resulted from rolling microdrops, as indicated by micro­scopic observation, were not included in the results. E. Experimental Conditions for Neural Stimulation of the Muscle The sens itivity of norma I neuromuscu lar transm iss ion to atropine and DTC was determined in the following manner. The obturator nerve was electrically 19 stimulated with a Grass 5-4 stimulator, a stimu Ius iso lation un it {Fein, H., unpublished}, and a pair of platinum-iridium wire electrodes. The stimulus voltage was twice that required to evoke maximum twitch tension; the frequency of stimulation was once every 2 sec. During the period of electrical stimulation the test drug was administered intravenously, and the effect of the agent on maximal twitch tension was recorded on a polygraph. III. RESULTS A. Fibril latory Activity of the Denervated Anterior Graci( is Muscle As j( lustrated in Figure 3A, fibri Ilation cons Isted of asynchronous, spon­taneous action potentials and uncoordinated, miniature contractions involving many fibers in the muscle. Upon examination of individual musc Ie fibers by means of intracellu lar electrodes, the rhythmically d ischarg ing fibri lIatory potentials appear to be similar to the action potentials of normal muscle (Figure 3B). The fibrillatory movements of the anterior gracilis muscle can be easily observed. Fibrillation, which is one of the main characteristics of denervated mammalian muscle, commenced in a few fibers as early as the second day following section of the motor nerve. From 5 to at least 30 days after denervation the preparations demonstrated extens ive fibri lIatory activity, and the few 40-day-denervated anterior gracilis muscles examined still exhibited marked fibrillation. B. Development of ACh-induced Contracture Changes in contracture threshold with time after denervation were determ ined by both the filter paper technic and intra-arterially administered ACh .. The contracture thresholds were measured at the two end-plate zones and at a non­end- plate area by the topical method in twenty-three denervated muscles (Figure 4A, B, and C) .. Then contracture thresholds were also determined by the intra-arterial administration of ACh in the direction of the heart by means of the contralateral external iliac artery (Figure 4D). In this manner, the unique topical method was compared with the more classical means of evoking denervation contracture. 21 Two days following denervation ACh appl ied topically anywhere on the muscle evoked a contracture; thereafter the ACh thresho Id of the graci I is musc Ie at both end-plate and end-plate-free reg ions decreased unti I a minimum was attained at 20 days after nerve section (Figure 4A, B, and C). Un I ike the resu Its obtained from the end -plate-free reg ions and the proximal end-plate zones, the data obtained from the distal end-plate zones exhibited extensive, unexplained variability (Figure 4B). Therefore, in subsequent experiments data were not collected from the distal end-plate zone. The rate of development of denervation supersensitivity, as indicated by a decrease in threshold to topical ACh, appears very similar at all three test sites on the musc Ie (Figure 4A, B, and C). In other words, there appeared to be a un iform development of denervation sensitivity to ACh over the entire surface of the muscle. The data obtained by intra-arterial administration are much different from those obtained by the topical application of ACh (Figure 4D). First, the denervated muscles were 1000 to 10,000 times less sensitive to parenterally administered ACh than to topically applied ACh. Secondly, the results obtained by the parenteral route of administration were more variable than those obtained from the proximal end-plate zones and the end-plate-free areas. 22 Thirdly, maximum sensitivity appeared in five days after denervation compared with the twe~ty days observed with the topical method. In order to estimate the magn itude of th e denervation supersens i tivi ty, three normal muscles were treated with topically and intra-arterially administered ACh (see filled circles in Figure 4A, B, and D). Since ACh does not produce a contracture in the normal anterior gracilis muscle, the threshold was redefined as the min imal amount of ACh required to evoke contraction. ACh appl ied to normal end-plate zones or administered intra-arterially to normal muscles elic ited a burst of action potentia Is and a brief contractile response (Figures 5A and 6A). In contrast, very high concentrations of ACh (5 x 10-1 ,.,g/fJ I) fai I ed to evoke any response from end-plate-free sites of normal muscle (Figure 6B). C. Effect of ACh on Extracellularly Recorded Fibrillatory Potentials In the twenty-three denervated animals utilized in the study of the in­fluence of 1'ime after denervation on the threshold for contracture, the effects of ACh on the fibrillatory potentials were examined. The results of the analysis can be readily summarized as follows. ACh applied to end-plate zones in increasing concentrations produced no change, an increase, an increase fo I lowed by a decrease, or a decrease in the fibrillatory potential rate. Some of the responses are seen in Figure 7A and B. In contrast, ACh applied to end-pfate­free sites on the muscle never increased the rate of the fibrillatory potentials and usually had no measurable effect on the conduction of these potentials (Figure 7C). As can be seen in Figure 7 A, B, and C, the above-described resu Its of ACh appl ication were produced by concentrations of the drug that elic it contracture. 23 In the same series of experiments on the development of denerva"'ion super­sensitivity, ACh was also administered intra-arterially. In the denervated muscles, ACh produced a burst of action potentials and a brief contractile response followed by a more prolonged contracture associated with a decrease or suppress ion of the fibri lIatory potentia Is (Figure 5B). In other words, ACh evoked an initial brief, propagated, electrical response and a biphasic, con­tracti I e response. D. Relationship Between Depolarization and the Concentration of Topically Applied ACh The data, as illustrated in Figure S, indicate that the magnitude of de­polarization in denervated preparations is quantitatively related to the con­centration of ACh topically applied by the microdrop method. The concentra­tions of drug needed to produce a response on the I inear portion of the concen­tration- depo arization curve compared favorably from one muscle to another if the volume of the drop and time after denervation were the same in each ex­periment. For example, the muscle used to obtain the data shown in Figure SA appears to be approximately ten times more sensitive than that used to obtain the results depicted in Figure SB. The apparent greater sensitivity can be ex­plained by the fact that a drop volume of 1 pi was used in the former experiment compared with 0.5 p' in the latter. In addition, it was observed that the maximum 24 response of different 20-day-denervated preparations varied from less than 1 mV in some muscles to lS mV in others. The maximum responses obtained from end­plate zones and end-plate-free areas of any given preparation differed by a few mV (Figure SA). In six denervated muscles the end-plate zones showed greater maximum sensitivity than did the end-plate-free areas; in five denervated preparations the reverse was true. In the determination of the relationship between depolarization and the concentration of ACh, solutions of varying concentrations were repeatedly applied to the same test site on the denervated muscle. Most of the test sites demonstrated tachyphylaxis or desensitization to ACh. Tachyphylaxis manifested itself as a decrease in depolarization at high concentrations of ACh (Figure SA). Similar concentration-depolarization curves were obtained by applying ACh to different sites on the membrane (Figure SB and C). By avoidance of repeated application of ACh to a specific site of the membrane, desensitization was prevented (Figure SB and C). Normal muscle frequently exhibited unpredictable depolarizations and hyperpolarizations in response to the top ica I appl ication of 1 .0 P I of 0.9'/0 NaCI, mammalian Ringer's, Locke's, or Krebs-Henseleit solution. Therefore, it was impossible to record reliably the local depolarizaHons produced by ACh at normal end-plate zones. In contrast, denervated muscles infrequently responded to topically applied O.CfO/o NaCI solution, and the few preparations that did respond were discarded. 25 E. ACh-induced Depolarization and Contracture The quantitative relationsh ip between ACh-evoked depolarization and the amplitude of contracture was determined at the proximal end-plate zone and at one end-plate-free site in six different denervated preparations by the micro­drop method. As illustrated in Figure 9D, depo larization a Iways preceded and outlasted contracture, and contracture never occurred without prior depolarization. In addition, above a threshold value the degree of ACh-evoked depolarization was proportional to the magn itude of the contracture (Figure 9A). The de­polarization ctnd tension relationships were similar at both end-plate-free regions and at end-plate zones (Figure 9A). In seven muscles denervated ,From 8 to 30 days, the effects of ACh on the transmembrane potential at an end-plate-free site of an individual muse Ie fiber were recorded concurrently with the ACh-induced tension changes of the entire muscle. The recording electrode was frequently dislodged from within a fiber as a result of fibril latory contractions or ACh-evoked contractureo Con­sequently, only twenty-eight different fibers were successfully examined in seven experiments. In twenty-three out of twenty-eight tests ACh produced both depo larization and contracture; in the other five tests, on Iy depolarization. In all the experiments depolarizations ranged from 7 to 63 mY and the contractures ranged from 20 to 215 mg. Of the twenty-three responses, depolarization preceded contracture in fourteen, contracture preceded depolarization in four, and both events occurred at the same time in the remaining five. 26 The magnitude of the contracture tended to be proportional to the degree of the depolarization; however, there was considerable variability within each experiment. In four different tests ACh yielded a depolarization of less than 20 mY and a marked contracture, and in five other tests ACh produced depolariza­tions ranging from 6 to 27 mY without producing a contractile responseo In the twenty-three ACh tests in wh ich depol arization and tens ion resu Ited, seventeen fibers were acHvely fibri lIa1"ing before the appl ication of the drug. Under these experimental conditions ACh did not increase the rate of discharge of the fibrillatory potentials, but it decreased the amplitude or abolished ,the fibri lIatory potentia I (Figure lOA). The study of the recovery of the membrane potential from ACh effects was hindered by the frequent ej ection of the electrode from the fiber by the contracture or by fibrillatory contractions. When the recording electrode remained within the muscle cell at the end of the contracture, the membrane was sti II markedly depolarized. In three fibers in wh ich complete recovery was almost attained, the membrane repolarized slowly toward normal values, and the fibrillatory potentials gradually began to reappear. In six of the twenty-three ACh tests that resu I ted in both depolarization and increased tension, the denervated muscle fiber was not fibrillating. Under these experimental conditions, ACh produced depolarizations as high as 60 mY and a contracture, but the marked depolarizations of the fiber produced by the drug did not result in the generation of an action potential (Figure lOB). 27 F. Local Nature of ACh -induced Responses A drop of ACh solution must surround the tip of the glass recording electrode in order for one to measure the depolarization of the denervated membrane. In some tests the ACh solution ejected from the microapplicator accidentally rolled away from the microelectrode, and the resulting depolarization was much less than anticipated or was not recorded at all. In order to demonstrate more clearly the local nature of the depolarization, the following experiment was performed. A drop of ACh was placed at the tip of the recording electrode and the resu Iting depolarization measured (Figure 11 A). After the first drop of ACh was removed from the muscle, an additional drop of drug solution was applied 3 mm away from the microelectrode, which did not detect, under such circumstances, any degree of depolarization (Figure 11 B). There are experimental findings indicating that ACh depolarization is not only local but also is non­propagated. For example, ACh markedly decreased the transmembrane potential at end-plate-free areas without evok ing an action potential (Figure lOA and B). G. Effects of d-Tubocurarine (DTC) In experiments in wh ich DTC was employed, each drop of ACh solution was placed on a different spot on the denervated muscle in order to avoid desensitiza­tion. The intravenous administration of 0.5 mg of DTC per kg shifted the ACh concentration-depolarization curve obtained at end-plate-free regions of three denervated muscles markedly to the right. As can be seen in Figure 8B, approximately 200 1'imes as much ACh was required to produce a given intensity 28 of depolarization after DTC as compared to the controls. Although DTC in-creased the concentration of ACh required to produce a given amount of depolariza­tion in an additional three denervated preparations, it did not alter the relation­ship between the degree of ACh-evoked depolarization and the magnitude of contracture (Figure 9B). In normal musc les O. 1 to 0.5 mg of intravenously adm in istered DTC per kg compl ete Iy blocked neuromuscu lor transm iss ion (Figu re 12A) • H. Effects of Atropine In the following series of experiments, as in those involving DTC, each drop of drug solution was appl ied to a different site on the denervated musc Ie in order to avoid desensitization. Intravenous administration of 0.5 rng of atropine per kg also markedly shifted the ACh concentration-depolarization curve obtained from end-plate-free regions in three denervated musc les. As indicated in Figure 8C, approximately 200 times the concen tration of ACh is requ ired to evoke a given intensity of depolarization after atropine as compared to the controls. AI though atropine increased the concentration of ACh requ ired to cause a given amount of depolarization in three other denervated preparations, it did not alter the relationsh ip between the extent of the ACh-produced depolarization and the magnitude of contracture (Figure 9C). A,tropine in doses ranging from 0.5 to 1.5 mg per kg completely abolished neuromuscular transmission in four normal muscles (Figure 12B). After a low dose of atropine, transmission recovered within 30 minutes, but with 1.5 mg per kg transmission failed to recover within two hours. 29 I. Effects of Norepinephrine and Isoproterenol The effects of norepinephrine and isoproterenol administered parenterally and applied topically by the microdrop technic were examined in eleven muscles 5 to 30 days following section of the motor nerve. One of the major character­istics of the effects of the catecholamines on the denervated anterior gracilis muscle is the inconsistency of the DC potential change and contracture. Therefore, no attempt was made to quantify the results, and the experimental findings are presented in terms of general tendencies observed in the experiments. The only consistent response to the topically appl ied catecholamines was an increase in the rate of the fibri Ilatory potentia Is. Microdrops of norepinephrine and isoproterenol (1 to 20 pg/fJ I) were appl ied to both end .... plate and non-end-plate reg ions of denervated muscle. Usually, there was no DC potential change or contractile effect of the drugs (Figure 13A and E) • However, they occasionally caused small hyperpolarizations and less frequently minute depolarizations. In all the experiments ACh evoked both a depolarization and contracture and O. <P/o NaCI solution had no effect (Figure 13B and F). In contrast to the top ica I adm in istration, intra-arteria I administration of 1 to 15 f1g of catecholamine per kg usually caused a depolarization and a contracture (Figure 13C and G). Tachyphylaxis quickly developed to the effects of the adrenergic agents and cross-tachyphylaxis with intra-arterially administered ACh frequently appeared to occur. Of the eleven preparations included in the 30 study, three were not responsive to the intra-arterial admin istration of the catechol­amines. Some experiments were also performed with epinephrine. The results ob­tained were similar to those described for norepinephrine and isoproterenol. IV. DISCUSSION The anterior gracilis muscle of the rat was chosen as the test object because it is uniquely suited for the investigaf"ion of end-plate-free sites. It is relatively simple to identify end-plate-free areas because the end-plates of this muscle in the rat are in two discrete, 1-mm bands (Figure 1), easi Iy located by anatomical and electrophysiological criteria (see METHODS). In contrast, the end-plates of most skeletal muscles are not located within a specific zone on the muscle but are widely distributed over the fibers (Tiegs, 1953). With a simple method for determining the end-plate-free regions and a micromethod for the local application of drug so lutions, the pharmaco log ica I properties of th e receptors at end-plate-free sites can be readily investigated. In most previous studies of denerva'l'ion supersensitivity, it was not possible to distinguish between responses of the end-plate and end-plate-free reg ions of the muscle because the test drugs were added to a bath ing solution or admin istered parenterally. Two prominen t characteristics of denervated musc les are ACh -induced contracture and spontaneous fibrillation, and one of the aims of the present work was to investigate the relaHonsh ip between fibrillation and contracture. In order to examine fibrillatory activity, it is necessary to perform the experiments in situ because fibrillation disappears rapidly in vitro (Thesleff, 1963; Eyzaguirre, C., u npub I ished) . An important benefit accru ing from the study of a denervated muscle in situ is that the fibrillatory activity serves as an excellent index of the physiological 32 status of the preparation. For example, the ampl itude and the frequency of fibrillatory potentials are very sensitive to changes in blood flow, body tempera­ture, and muscle stretch (H~k & Skorpil, 1962; Belmar & Eyzaguirre, 1966). Therefore, the character of the fibrillatory activity provides a reference for the functional state of the muscle. Fibrillation consisted of spontaneous action potentials and miniature con­tractions (Figure 3) and generally appeared maximal within 5 days after section of the motor nerve. Jarcho, Berman, Eyzaguirre, & Lilienthal (1951) reported similar observations on the anterior gracilis of the rat. Luco & Eyzaguirre (1955) noted that the time of onset of fibrillation is a function of the length of the distal segment of the degenerating nerve stump. Therefore, in the present investigation an attempt was made to produce distal nerve stumps of approximately equal length. The principal purpose of the present work was to study some of the properties of muscle associated with denervation supersensitivity. Previous investigators demonstrated that the complete manifestation of denervation sensitivity to ACh requires time (Brown, 1937; Luco & Rosenblueth, 1937; Axelsson & Thesleff, 1959; Miledi, 1960a); consequently, it was necessary to determine the time course for the development of supersensitivity in the anterior gracilis muscle. In the present work the development of supersensitivity was measured in terms of ACh­contracture threshold (Figure 4). The sensitivity of the muscle to ACh progressively increases and reaches a maximum 20 days after denervation. Most of the subse­quent experiments involving ACh supersensitivity were conducted on animals 33 denervated for a period of 20 to 25 days in order to standardize the preparations at a time adequate for development of maximum sensitivity. The contracture-threshold data obtained during the study of the development of supersensitivity with time illustrate several pharmacological properties of the denervated anterior gracll is muscle. For example, ACh produced a contracture in denervated muscle regardless of 'the site of application (Figure 4A, B, and C). In the normal muscle ACh can elicit a twitch only upon application to end-plate zones (Figure 6). The data of Ginetzinsky & Shamarina (1942), Axelsson & Thesleff (1959), Miledi (1960a) and Elmqvist & Thesleff (1960) suggest that the newly-formed sensitive area deve lops first in the reg ion surround ing the end-plate and with time slowly extends from the end-plate region towards the muscle area near the tendon. These observations on the spread of supersensitivity are not supported by the anterior gracilis muscle experiments which indicate a uniform development of sensitivity over the entire muscle. A possible explanation for the discrepancy in the observations is the fact that the various experiments involved different muscles and different species. The size of the filter paper employed for the topical application of ACh limits the interpretation of the data obtained from end-plate zones. The appl ica­tion of ACh with 3-mm squares of filter paper to end-plate areas does not dis­criminate between the end-plates themselves and the end-plate-free membrane with in the end-plate zone. The end-plates per se comprise a relatively small 34 area compared to the total area of the end-plate zone. In addition, the filter paper is wider than the end-plate zone. Therefore, data obtained from end-plate r~ions may reflect mainly, if not only, the sensitivity of end-plate-free membrane. The resu Its obtained concerning the development of denervation sensitivity cannot be interpreted to mean that the end-plates become supersensitive. Axelsson & Thesleff (l959) and Thesleff & Elmqvist (l960) reported that the sensitivity of an end-plate to iontophoretically applied ACh does not change following denervation.. They cone luded that on Iy the end-plate-free area of the membrane changes in sensitivity, and at maximum development of sensitivity the end-plate-free portion of the denervated mammal ian muscle membrane approaches the sensitivity of a normally innervated end-plate. All the data considered in the subsequent part of this DISCUSSION were obtained with the microdrop technic. The method was developed in order to circumvent some of the disadvantages associated with the filter-paper method .. The problems of the filter-paper method that are avoided by the use of the microdrop technic include the following: variability in the amount of drug solution applied, production of electrical and mechanical artifacts associated with the appl ication of the filter paper to the muscle, and interference with the recording of local DC potential changes. The size of the m icrodrop, I ike the size of the fi I ter paper, is important in the interpretation of the data. The measured diameter of a 1-,., I drop surrounding the electrode tip is approximately 1 mm and that of a O. 51J I 35 drop is approximately 0.5 mm. The smaller size of the drop compared with the size of the filter paper greatly facilitated the srudy of end-plate-free reg ions. The small size of the microdrop increased the potential number of different test sites on the membrane by an order of magnitude. The drop size, however, was still far greater than the size of an individual end-plate; conse­quently, the characteristics of the end-plate receptors themselves could not be studied. The same problem of overlap of solution on end-plate and end-plate­free areas exists as described above for the filter-paper method. It is known that ACh produces both depolarization and contracture in the denervated muscle (Elmqvist & Thesleff, 1960). Most investigators consider that in normal muscle the initial step leading to contraction is the reduction of the membrane poteni"ial (Kuffler, 1946; Katz, 1950; Sten-Knudsen, 1954, 1960; Huxley & Taylor, 1958; Watanabe, 1958; Hodgkin & Horowicz, 1960; Orkand, 1962). Whether such coupl ing exists in the denervated muscle stimu lated by ACh is not known. Jenkinson & Nicholls (1961) observed that ACh evoked a contracture in potassium-depolarized denervated muscle without any concomi­tant depolarization. The possib il ity, therefore, exists that depolarization of denervated preparations is the consequence of an interaction of ACh and newly­formed receptors that are not electrically linked to the contractile mechanism. The data shown in Figure 8A suggest a quantitative relationsh ip between the concentration of ACh and the depolarization. A similar quantitative relation­ship appears to exist between the concentration of ACh and the depolarization 36 at end-plqte zones and at end-plate-free regions. As discussed earl ier, however, end-plate tests probably refl ect on Iy the character of denervated end-plate-free membrane. Another characteristic of the newly-formed receptors is the development of desens itization or tachyphylaxis to repeated appl ications of ACh to a spec ific test site. The desensitization is clearly seen by the decrease in depolarization produced by high concentrations of ACh, as illustrated in Figure 8A. In the eleven concentration-effect studies there was some variation in the maximum depolarization obtainable at the two separate test sites with in one experiment, and the variabil ity may be explained by differences in the onset of tachyphylaxis. The fact that desensitization of cholinergic receptors in denervated and normal muscle can occur has been reported previously (Thesleff, 1955; Katz & Thesleff, 1957; Axelsson & Thesleff, 1959). To study the electrical-mechanical relationships in the denervated muscle, ACh-induced depolarizations and contractures were simultaneously measured. A qual itative examination of extracellularly-recorded ACh responses, such as those in Figure 9D, suggests that there is a causal relationsh ip between depolariza­tion and tension • First, depolarization always preceded and outlasted the con­tractile response; secondly, contracture did not occur without prior depolariza­tion; and thirdly, as the amount of depolarization increased, the magnitude of the contracture increased. The quantitative relationsh ip between depolarization and contracture is 37 shown in Figure 9A, S, and C. The magnitude of the contracture is directly proportional to the degree of depolarization. Similar results were obtained at both end-plate and end-plate-free reg ions (Figure 9A). The qua Ii tative and quantitative relationsh ips between ACh-induced depolarization and contracture suggest the existence of coupling between electrical and mechanical events. An attempt was made to study the relationship of depolarization and con­' tracture by simultaneously measuring ACh-induced changes in 'transmembrane potential and tension. Although the resu Its suggest the possibi I ity of a causal relationship, inconsistent results reflected serious limitations in the approach. For example, contracture occasionally occurred before depolarization, and there was marked variabi I ity in the quantitative relationsh ip between the extent of depolarization and the development of tension. Despite these inconsistencies, depolarization generally preceded contracture, and the tension developed tended to be related to the degree of depolarization. From the studies discussed above it was obvious that the extracellu lar re­cording techn ic provided more consistent results than did the intracellu lar method. The difficulties with the intracellu lar recording procedure appear to arise from the fact that the measured electrical activity is the product of a sing Ie fiber, whereas the measured tension is produced by many fibers. Therefore, fibers which are protected by several layers of cells may be depolarized later than those on the surface of the muscle, and it is conceivable that contracture can precede depolarization. Furthermore, deeply situated cells may receive less 38 ACh and may depolarize less than other fibers contributing to the contracture, and consequently the quantitative relation between depolarization and tension may be distorted. An additional complication with the intracellu lar studies resulted from the difficulty in maintaining even a floating electrode within a fiber during spontaneous fibrillatory movements and an ACh-evoked contracture. Finally, the limited number of cells successfully studied may represent a select and non-representative sample of the total popu lation. Kuffler (1946), Huxley & Taylor (1958), Hodgkin & Horowicz (1960), and Orkand (1962) concluded from studies of single-fiber preparations of normal frog, crab, and crayfish muscle that depolarization is causally related to the tension developed. These observations in single-fiber preparations represent some of the best evidence supporting the concept that depolarization and tension are related in normal muscle. Unfortunately, similar single-fiber experiments on denervated preparations and norma I mamma I ian musc I es have not yet been described. The data available suggest that ACh-evoked depolarization in the denervated anterior gracilis muscle is linked to contracture. Such a conclusion does not conflict with the observation of Jenk inson & Nicholls (1961) that ACh can produce contracture in the potassium-depolarized, denervated rat diaphragm. There are several unusual properties of the contracture elicited in the potassium­depolarized muscle. First, in comparison with an untreated denervated diaphragm, the potassium-depolarized denervated preparation requires thirty to fifty times 39 more ACh to produce a minimal contracture. Secondly, the maximum contracture obtained in the potassium-depolarized denervated muscle represents approximately only 20 per cent of the maximum contracture of the control denervated diaphragm. Thirdly, the depolarized denervated muscle appears to be more sensitive than the control denervated muscle to a decrease in calcium concentration in the bathing medium. The last and most unusual property is that the contractures in po tass ium-depolarized muscle are extremely temperature-dependent. For example, the responses usually occur between 18 to 220 C and do not occur above 300 C. In contrast, the experimental findings with the anterior gracilis muscle of the rat were obtained at normal body temperatures. In some of their classical investigations Brown (l937} and Rosenblueth & Luco (1937) demonstrated the effects of intra-arterially administered ACh on mammalian skeletal muscle. They noted that in normal muscle ACh produced a burst of action potentials as well as a few brief twitches; they referred to the combination of events as the uquick phase" or the "contraction. If In denervated muscle ACh yielded an initial "quick phase" followed by a more prolonged in­crease in muscle tension called contracture. Higher doses of ACh in denervated muscles produced a similar biphasic response, but the initial contraction phase was followed by an abrupt suppression of all action potentials. The "electrically­silent" period associated with high doses of ACh frequently outlasted the con­tracture, and additional ACh during the silent phase evoked only a prolonged contracture. In the present work with the anterior gracilis muscle, intra-arterially administered ACh elicited all the effects described above; responses of normal and denervated muscle to ACh are illustrated in Figure 5. Some of the effects of ACh appl ied to end-plate zones of the anterior gracilis muscle resembled those produced by the intra-arterial administration 40 of ACh . For example, the appl ication of the drug to the end-plates of norma I muscle resulted in a short burst of action potentials and a few twitches (Figure 6A). In denervated muscle ACh appl ication to the end-plate area produced no change, an increase, an increase followed by a decrease, or a decrease in the fibri lIatory potential rate, as well as a prolonged contracture. Some of the responses are shown in Figure 7A and B. Unlike parenteral administration, local application of the drug did not cause a biphasic contractile response. Local application of ACh to end-plate-free regions of the muscle provided markedly different results. ACh applied to normal muscle did not evoke an effect, even in heroic concentrations of 5 x 10-1 fg/P I (Figure 6B). ACh caused a contracture of denervated muscles without changing the rate of the fibri lIatory potentia Is (Figure 7C). An investigation of the effects of ACh at end-plate-free reg ions by means of intracellu lar recording methods demonstrated that drug concentrations pro­duc ing contracture reduced the membrane potential difference, reduced the amplitude of the fibrillatory potentials, and frequently abolished the fibrillatory potentials of the test muscle fiber (Figure lOA). Using both intracellular and extracellular recording technics, Belmar & Eyzaguirre (1966) demonstrated that 41 ACh increased the rate of the fibri lIatory potentials of the denervated anterior grac His musc Ie of the rat on Iy upon its appl ication to the end-plates. The effects of topical ACh on the anterior gracilis muscle can be summarized as follows. ACh applied to the normal muscle evokes a contraction only when applied to the end-plate zones (Figure 6); however, ACh evokes a contracture in denervated muscle when applied anywhere on the membrane (Figure 4A, B, and C). The drug increases the rate of the fibrillatory potentials of denervated muscle when applied to end-plate zones but not when applied to end-plate-free sites (Figure 7A and C). Belmar & Eyzaguirre (1966) reported the results of a quantitative investigation of the effects of ACh on fibri Ilatory potentials of the denervated anterior gracilis of the rat. Their results of experiments on denervated end-plate zones indicate that an ACh concentration of 10-6 pg/,.,I increases the fibri Ilatory potential rate, whereas a concentration of 10-5 pg/p I decreases the rate. In addition, ACh applied to end-plate-free sites usually had no effect on the extracellu larly recorded spontaneous potentials. The present and previous studies indicate that ACh increases the rate of the fibrillatory potentials only upon appl ication to a denervated end-plate zone. However, ACh is capable of producing a contracture anywhere on the denervated muscle membrane. Thus, it can be concluded that contracture is not the result of a massive discharge of fibrillatory potentials. Furthermore, fibrillation rapidly ceases in vitro (Thesleff, 1963) without affecting ACh-induced contracture; hence, the contracture characteristics of denervated muscle do not appear to be dependent on propagated potentials. tf' ECCLES HEAL TM SCI 42 In normal muscle, contraction is the consequence of a propagated depolariza­tion of the membrane. The present work on the denervated anterior grac iI is muscle indicates that depolarization produced by the topical appl ication of ACh is local in nature (Figure 11) and not propagated. In further support of the view that depolarization is not propagated is the observation that ACh applied to end-plate-free sites markedly depolarizes non-firing muscle cells but does not produce an action potential (Figure lOB). The depolarized part of the membrane probably consists of the area of membrane in actual contact with the drug solution and an additional area determined by the length constant of the mem­brane. Since the depolarization and the contractile responses appear linked, contracture is probably restricted to the depolarized region of the membrane. If one observes contractures under a microscope, they do appear to be limited to the site of appl ication of the drug solution. Kuffler (1946) and Sten-Knudsen (1954) agree that, in normal muscle, contracture is confined to the region where the membrane potential is reduced. The observations of Elmqvist & Thesleff (1960) provide further support for the interpretation that the response is local in nature. They reported that the magnitude of the contracture elicited by ACh in the denervated rat diaphragm preparation in vitro is a function of the size of the sensitive area of the membrane and the length constant of the muscle. Gasser (1930), in his classical review, defined contracture in both normal and denervated muscle as a local, non-propagated, prolonged contractile response associated with a DC potential change. The more recent studies 43 described above lend additional support to Gasser's original definition. The concept that the transmitter-sensitive area of skeletal muscle enlarges after denervation is well established (Axelsson & Thesleff, 1959; Miledi, 1960, 1962). In addition, it is known that the ACh responses of denervated muscle are selectively qntagonized by curare (Gasser & Dale, 1926; Brown, 1937; Luco & Rosenblueth, 1937; Jarcho, Berman, Eyzaguirre, & Lilienthal, 1951; Bhoola & Schacter, 1961). Extremely high concentrations of atropine are required to oppose the ACh-evoked contractures of denervated amph ibian and mammalian muscle (Gass.,r & Dale, 1926; Dale & Gaddum, 1930), and muscarinic agents such as pi locarpine and muscarine do not stimu late denervated musc Ie (Dale & Gasser, 1926). From these observations it has been assumed that the newly-formed chol inergic receptors are n icotin ic in character. In the above-cited studies of the pharmacological nature of cholinergic receptors, the agonists and antagonists were added to a bathing solution or administered parenterally. Under these classical experimental conditions the responses of receptors on a specific part of the membrane are not distinguishable. In only a few previous investigations have the receptors at the end-plate zones and the end-plate-free sites on the muscle been separately examined. For example, a comparison of the sensitivity of various regions of mammalian de­nervated muscles to iontophoretically applied ACh suggested that responses of all the cholinergic receptors are very similar (Axelsson & Thesleff, 1960; Elmqvist & Thesleff, 1960). Jenkinson (1960), using DTC, showed the nicotinic character of the receptors of denervated frog muscle. Berbnek & Vysko~iI (l967), utilizing DTC and atropine, demonstrated the nicotinic nature of the receptors at both end-plate and end-plate-free areas of the denervated rat diaphragm. 44 In contrast to previous descriptions of the newly-formed receptors, experi­mental findings of the present investigation indicate the development of muscarin ic as well as nicotinic receptors on the end-plate-free regions of the denervated anterior gracilis (Figure 8B and C). DTC is known to antagonize selectively the effects of ACh on denervated musc Ie; consequently, the existence of n icotin i c receptors at end-plate-free sites of the membrane is not surprising. It is difficult to assume that all mammalian denervated muscles develop muscarinic receptors because relatively high concentrations of atropine are required to antagonize the effects of ACh on some denervated mammal ian muscles (Dale & Gaddum, 1930; Berbnek & Vyskog i I, 1967)_ The anterior gracilis muscle may in fact be a unique skeletal muscle because of the demonstrated ability of atropine to block selectively neuromuscular trans­mission in normal preparations (Figure 12B). Therefore, it appears that the newly­formed muscarinic and nicotinic receptors are an extension of the normal character of the muscle. The cholinergic receptors of the rat diaphragm are nicotinic before and after denervation (Bera'nek & Vysko~iI, 1967). As observed in the current study, the receptors of the rat anterior gracilis muscle are muscarinic and nicotinic both before and after denervation. The resu Its suggest that the nature of cholinergic receptors of normal and denervated muscle is mainly de­termined by the muscle rather than by the nerve. From the present work, it is impossible to note slight differences in the character of the receptors; some sl ight alteration in the nicotinic receptors following denervation has been suggested by some investigators (Jenk inson, 1960; Ber~nek & Vyskog ii, 1967). 45 The experiments with DTC and atropine provide additional support for the conclusion that depolarization and contracture are linked. In the experimental findings shown in Figure 9B and C, the concentration of ACh required to produce a given amount of depolarization was markedly increased after the parenteral administration of DTC or atropine, but the quantitative relationsh ip between depolarization and contracture did not change. Although these observations do not provide definite proof, the results suggest that ACh is not acting on two independent receptor systems, such as one on the membrane and the other in the contracti Ie system. Denervated muscles also become supersensitive to epinephrine, norepinephrine, and isoproterenol administered intra-arterially (von Euler & Gaddum, 1931; Balbring & Burn, 1936; Luco & Sanchez, 1956; Bowman & Zaimis, 1961), and parenterally administered ~-adrenergic blocking drugs antagonize the effects of these agents on denervated"iTfusc Ie (Bowman & Raper, 1965). Since super­sensitivity to cholinergic agents results from the development of new cholinergic receptors on the membrane (Axelsson & Thesleff, 1959; Miledi, 1960), it is reasonable to assume that supersensitivity to catecholamines results from a 46 similar development of new adrenergic receptors. The experiments designed to delineate the pharmacological characteristics of the adrenergic receptors of the denervated membrane were generally unsuccessful. Topically applied catecholamines did not produce depolarization and contracture (Figure 13Aand E), despite the fact that parenterally administered catecholamines produced both depolarization and contracture (Figure 13C and G). Brown, Go ffa rt , & Vianna Dias (l950) studied the effects of parentera lIy admin istered catecholamines on the demarcation potential and on the tension of the denervated cat tibialis muscle. They observed that the catecholamines produced an initial small hyperpolarization followed by a marked depolarization, as well as a prolonged contracture. Maximum contracture occurred at the peak of hyperpolarization and relaxation occurred during depolarization. Bowman & Raper (1965) studied the same muscle preparation as did Brown et al., but they reported that parenterally administered catecholamines produced either small hyperpolarizations associated with contractures or large hyperpo larizations assoc iated with relaxation. There is no obvious explanation for the discrepancies in these studies of denervated skeletal muscle. Although the topical appl ication of catecholamines to the denervated anterior gracilis muscle did not elicit a contracture, the adrenergic agents stimulated the rate of the fibrillatory potentials. Thus, it appears that the only adrenergic receptors on the membrane of the denervated gracilis muscle are those associated with the fibrilfatory potentials. 47 Some general conclusions can be drawn from the findings of the experiments with catecholamines. First, the results suggest that intra-arterially administered adrenergic drugs reach a site of action which is unobtainable by the topical route of administration. Secondly, there appear to be adrenergic receptors on the surface of the denervated muscle membrane, but they are only associated with the fibri lIatory potentials. Therefore, un I ike ACh denervation super­sensitivity, it is difficult to explain the exaggerated response to the catechol­amines following nerve section by the development of receptors over the entire surface of the muscle, Bhoola & Schacter (1961) reported that epinephrine caused contracture of the denervated rat diaphragm in vitro. It wou Jd be interesting to re-investigate the effects of adrenergic agents on the denervated rat diaphragm and other muscles with the microdrop technic in order to compare the results with those of the current investigation. The data concerning the effects of the catecholamines support a conclusion made above, that contracture is not the result of an increased rate of discharge of the fibri lIatory potentials. Catecholamines appl ied topically produced a marked increase in the fibrillatory potential rate and failed to evoke a contracture. Zaimis & Bowman (1961) were unable to correlate the effects of adrenergic agents on the fibrillatory potentials with those on the contractile system of denervated cat muscle. They also concluded that the fibrillatory potential rate is not related to contracture of denervated muscle. However, Bowman & Raper (1965) reported that the previously published results of Zaimis & 48 Bowman (1961) were in error and that denervation contractu re produced by parenterally administered catecholamines was the result of an increase in the fibrillatory potential rate. The results of the present investigation are in conflict with the conclusions of Bowman & Raper. Catecholamines topically applied to the denervated anterior gracilis muscle markedly increased the fibrillatory potential rate and did not produce a contracture. Intra-arterially administered catecholamines increased the fibri lIatory potential rate and produced a contracture. However, the occurrence of two parallel events does not necessarily indicate a cause and effect relationship. In the present inves'tigation the effects of catecholamines on the fibri Ilatory potential rate and on the contractile mechanism have been completely separated. Therefore, it appears that denervation contracture evoked by the catecholamines is not the consequence of an increase in the fibri lIatory potentia I rate. V. SUMMARY The physiological and pharmacological properties of the end-plate-free sites of the denervated anterior graci I is musc Ie of the rat were studied by means of a new microdrop technic for the topical application of drug solutions. ACh sensitivity after denervation developed at a uniform rate over the entire surface of the musc Ie. Such a conclusion is in confl ict with current con­cepts that ACh sensitivity spreads from the end-plate area towards the muscle area near the tendon. Depolarization is quantitatively related to the concentration of topically appl ied ACh, and the degree of depo larization is proportional to the magn itude of contracture. The prolonged denervation contracture of ACh-stimu lated muscle is not the consequence of a massive discharge of fibrillatory potentials, but is a non-propagated response produced by a persistent, local depolarization. The anterior gracilis muscle is a pharmacologically unique skeletal muscle because the junctional receptors of the normal muscle and the newly-formed receptors on end-plate-free regions of the denervated muscle are muscarinic as well as nicotinic in character. Thus, the ACh-induced contracture of the anterior gracilis muscle after denervation is the result of the development of both muscarinic and nicotinic receptors over the entire surface of the muscle. In contrast to the effects of ACh on the denervated muscle, catecholamines applied topically did not produce either depolarization or contracture. However, catecholamines administered intra-arterially produce both depolarization and 50 contracture. The only adrenerg ic receptors found on the surface of the denervated muscle membrane are those associated with the fibrillatory potentials. Unl ike ACh-induced contracture, it does not appear that catecholamine-induced con­tracture of denervated muscle is the result of the development of new adrenergic receptors on the membrane. VI. REFERENCES Axelsson, J. & Thesleff, S. (1959). A study of super sensitivity in denervated mammalian skeletal muscle. J. Physio!. 147,178-193. Bain, W. A. (1932). On the mode of action of vasomotor nerves. J. Physiol. 77, 3-4P. Belmar, J. & Eyzagui rre, C. (1966). Pacemaker site of fibri lIation potentials in denervated mammalian muscle. J. Neurophysiol. 29, 425-441. Bender, M. B. (1938). Fright and drug contractures in denervated facial and ocular muscles of monkeys. Am. J. Physiol. 121,609-619. Ber~nek, R. & Vysko~iI, F. (1967). The action of tubocurarine and atropine on the normal and denervated rat diaphragm. J. Physiol. 188, 53-66. Bhoola, K. 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Heidenhain, R. (1883). Ueber pseudomotorische Nervenwirkungen. Arch. f. Physio!., Leipz. Suppl., 133-177. Cited by Cannon, W. B. & Rosenblueth, A. ( 1949). The supersensitivity of denervated structures, pp. 6-7. New York: The Macmi Ilan Company. ,. Hnik, P. & Skorpil, V. (1962). Fibrillation activity in denervated muscle. The denervated muscle, ed. Gutmann, E., pp. 136-146. Prague: Publishing House of the Czechoslovak Academy of Sc iences. Hinsey, J. C. & Cutting, C. C. (1933). The Sherrington phenomenon. Am. J. Phys io I. 105, 535-546. Hodgkin, A. L. & Horowicz, P. (1960). Potassium contractures in single muscle fibres. J. Physio!. 153, 386-403. Hubbard, S. J. (1963). The electrical constants and the component conductances of frog skeletal muscle after denervation. J. Physiol. 165, 443-456. Huxley, A. F. & Taylor, R. E. (1958). Local activation of striated muscle fibres. J. Phys io I. 144, 426-441 . 54 Jarcho, L. W., Berman, B., Eyzaguirre, C., & Lilienthal, J. L. (1951). Curarization of denervated muscle. Ann. N. Y. Acad. Sci. 54, 337-346. Jarcho, L. W., Eyzaguirre, C., Berman, B., & Lilienthal, J. L. (1952). Spread of excitation in skeletal muscle: some factors contributing to the form of the electromyogram. Am. J. Physiol. 168, 446-457. Jenkinson, D. H. (1960). The antagon ism between tubocurarine and substances which depolarize the motor end-plate. J. Physiol. 152, 309-324. Jenkinson, D. H., & Nicholls, J. G. (1961). Contractures and permeability changes produced by acetylcholine in depolarized denervated muscle. J. Physio!. 159, 111-127. Katz, B. {1950}. Quoted by Hill, A. V. in A discussion on muscular contraction and relaxation: their physical and chemical basis. Proc. Roy. Soc. B, 137, 45-47. Katz, B. & Thesleff, S. (1957). A study of 'desensi1"ization ' produced by acetylcholine at the motor end-plate. J. Physiol. 138, 63-80. Kiku-i ri, T. (1964). Dissoc iation of el ectrical and mechanical events in de­nervated frog skeletal muscle. Jap. J. Physiol . .!.i,400-410. Koelle, G. B. (l95l). The elimination of enzymatic diffusion artifacts in the histochemical localization of cholinesterases and a survey of their cellular distributions. J. Pharmac. expo Ther. 103, 153-171. Koel Ie, G. B. (1955). The histochemical identification of acetylchol inesterase in chol inergic, adrenerg ic, and sensory neurons. J. Pharmac. exp. Ther. 114,167-184. Kuffler, S. W. {l943}. Specific excitability of the end-plate region in normal and denervated muscle. J. Neurophysiol. ~, 99-110. Kuffler, S. W. {1946}. The relation of electrica I potential changes to con­tracture in skeletal muscle. J. Neurophysiof. ~, 367-377. Langley, J. N. (1905). On the reaction of cells and of nerve-endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and curare. J. Physiol. 33, 374-413. Langley, J. N. (1921)_ The autonomic nervous system, pp. 80. London: Oxford University Press .. Cited by Cannon, W. B. & Rosenblueth, A. (1949). The supersensitivity of denervated structures, pp. 7-8. New York: The Macm i lion Company. 55 Luco, J. V. & Eyzaguirre, C. (1955). Fibrillation and hypersensitivity to ACh in denervated muscle: effect of length of degenerating nerve fibers. J. Neurophysiol. ~, 65-73. Luco, J. V. & Sanchez, P. (1959). The effect of adrena I ine and noradrenal ine on denervated skeletal muscle: antagonism between curare and adrenaline- I ike substances. Curare and curare-I ike agents, ed. Bovet, D., Bovet-N itti, F., & Marini-Bettolo, G. B., pp. 405-408. Amsterdam, London, & New York: Elsevier Publ ishing Company. Miledi, R. (1960a). The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation. J. Physiol. 151, 1-23. Mi ledi, R. (196Ob). Properties of regenerating neuromuscu lor synapses in the frog. J. Phys iol. 154, 190-205. Miledi, R. (1962). Induction of receptors. Enzymes and drug action, ed .. Mongar, J. L. & de Reuck, A. V. 5., pp. 220-235. Boston: Little, Brown, & Company. Nicholls, J. G. (1956). The electrical properties of denervated skeletal muscle. J. Phys io I. 131, 1-12. Orkand, R. K. (1962). The relation between membrane potential and contraction in single crayfish muscle fibres. J. Physio!. 161, 143-159. Philipeaux, J. M. & Vulpian, A. (1863). Note sur modification physiologue qui se produit dan Ie nerflingual par suite de "abolition temporaire de 10 motricit~ dans Ie nerf hypoglosse du m~me c'S't~. C. R. Acad. Sci. 56, 1009-1011. Cited by Cannon, W. B. & Rosenblueth, A. (1949). The supersensitivityofdenervated structures, pp. 6. New York: The Macmillan Company. Rosenblueth, A. & Luco, J. V. (1937). A study of denervated mammalian skeletal muscle. Am. J. Physiol. 120,781-797. 56 Sherrington, C. S. (1894). On the anatomical constitution of nerves of skeletal muscle; with remarks on recurrent fibres in the ventral spinal nerve-root. J. Phys iol. ,!Z, 211-258. Sten-Knudsen, O. (1954). The ineffectiveness of the 'window field' in the initiation of muscle contraction. J. Physiol. 125, 396-404. Sten-Knudsen, O. (1960). Is muscle contraction initiated by internal current flow? J. Physiol. 151, 363-384. Thesleff, S. (1955). The mode of neuromuscular block caused by acetylcholine, nicotine, decamethonium, and succinylcholine. Acta physiol. scand. 34, 218-231. -- Thesleff, S. (1963). Spontaneous electrical activity in denervated rat skeletal muscle. The effect of use and disuse on neuromuscular functions, ed. Gutmann, E. & Hn/ik-, -P~.,- p-p-. -41~-~51~. ~A-m-s-te-rd~a-m-, ~Lo-n-do~n-, -& ~N~ew~ ~Y~or-k-: Elsevier Publ ishing Company. Tiegs, O. W. (1953). Innervation of vol untary muse Ie. Physiol. Rev. 33, 90-144. Watanabe, A. (1958). Initiation of contraction by transverse and longitudinal current flow in single muscle fibers. Jape J. Physiol. 8,123-137. 57 Figure 1. The anterior graci I is muse Ie of the rat. A. Schematic representation of the muscle. a, the obturator nerve; b, the artery and vein serving the muscle; c, the proximal end-plate zone; d, the distal end-plate zone; e, the saphenous vessels and nerve passing over the muse Ie; f, the tendon near th e knee. B. Photomicrograph of the proximal end-plate zone of a bundle of normal fibers stained for cholinesterase by the thiochol ine technic. C. Photomicrograph of the proximal end-plate zone of two normal fibers stained in the same manner as in B. .... 111111111I 59 Figure 2. The microapplicator. A schematic representation of the device employed for the topical application of microdrops of solution to the muscle. A, a Hamilton syringe; B, a Hamilton PB600-1 repeating dispenser; C, a fine-bore polyethylene tubing; D, a blunted 27-gauge hypodermic needle. 61 Figure 3. The spontaneous activity of the denervated anterior gracilis muscle. A. Recording obtained from a 20-day-denervated muscle. The upper trace illustrates asynchronous spontaneous fibrillatory potentials produced by a number of fibers and recorded extracellu larly with platinum-iridium electrodes. The lower trace denotes the uncoordinated fibrillatory contractions. B. Recording obtained from a 21-day-denervated preparation. The upper trace illustrates the rhythmically-firing fibrillatory potentials of a single muscle fiber recorded intracellu larly with a conventional glass micro­el ectrode. The lower trace, as in A, denotes the uncoordinated fibrillatory contractions. (In both A and B the action potentials were slightly retouched for the purpose of clarity in photograph ic reproduction.) A 60 )JV 50mg B 60mV 50mg 15 sec 15 sec 63 Figure 4. The effects of time after denervation upon the ACh-contracture threshold of the anterior gracilis muscle. The open circles represent the ACh-contracture thresholds of denervated muscles. The filled circles illustrate the minimal doses or concentrations of ACh required in normal muscles to produce a burst of action potentials and contraction. All the circles and vertical bars denote the mean and ranges of the thresholds. Each threshold value is based on observations from three different muscles, except that the values for the 5-day-denervation thresholds are the mean of the results from five different muscles. Concentrations and doses of ACh are expressed as the log. A, B, and C. The data depicted were obtained with the filter paper technic. D. The data illustrated were obtained by the intra-arterial administration of ACh. ..t::: U « ;; 10-4 ...J o ::x: en I.LI ~ 10-5 to- I.LI D:: :::l t; 10.6 « 0:: to­Z 8 10-7 PROXIMAL END-PLATE ZONES TOPICALLY APPLI ED ACh 5 10 15 20 25 30 DAYS AFTER DENERVAT ION EN 0- PLATE - FREE AREAS TOPICALLY APPLIED ACh 10-8~--~--~~--~--~--__ ---TI 5 10 15 20 25 30 DAYS AFTER DENERV A T ION ..t::: U « ;; 10-4 ...J o X en LtJ ~ 10-5 to- LtJ D:: ::::> t 10-6 « D:: to­Z 8 10-1 D 1.0 1 0 ~ 0 e . u..t: :: 0.1 « 0 ...J 0 X CI) UJ D:: X to- I.LI 0.01 D:: :::l to-t..) « D:: to- Z 0 u 0.001 I I , , \ I I I , I , I , , DISTAL END-PLATE ZONES TOPICALLY APPLIED ACh -, 5 I (j 15 20 25 30 DAYS AFTER DENERVA T ION INTRA-ARTERIALLY ADMINISTERED ACh 5 10 15 20 25 30 DAYS AFTER DENERVA TlON 65 Figure 5. The effects of intra-arterially adm inistered ACh on the anterior grac iI is musc Ie. The action potentials were recorded by means of extracellu lar electrodes on the surface of the muscle. A. The responses produced in a normal muscle by the intra-arterial admin istration of ACh. The upper trace depicts a burst of action potentials, and the lower trace denotes the resu Iting contraction. B. The responses in a 20-day-denervated musc Ie evoked by the intra­arterial administration of ACh. The upper trace illustrates an initial burst of action potentia Is followed by depress ion of the fibri lIatory potentia Is. The lower trace demonstrates the typical biphasic contractile response, an initial contraction followed by a prolonged contracture. A 75)JV I 15 sec B 67 Figure 6. The effects of ACh topically applied to a normal anterior gracilis muscle. The action potentials were recorded with extracellular electrodes on the surface of the muscle, and ACh was applied by the filter-paper technic. A. The responses were elicited by the application of ACh to a normal proximal end-plate zone. The upper trace illustrates a burst of action potentials; the lower trace depicts the resu Iting muscle twitches. B. The appl ication of ACh to an end-plate~free reg ion of the norma I muscle failed to produce either an electrical or a contractile response. A 15pvl __ _ 15 sec 600 mg I __ -___ UL.."---------- 4 ACh I X lO-2)1g/pl B + ACh 5XIO-l pg / p l 69 Figure 7. The effects of ACh topically appl ied to denervated anterior gracilis muscles. The action potentials were recorded with extracellular electrodes on the surface of the muscle; ACh was applied by the filter-paper technic. (The action potential tracings were retouched for photographic reproduction.) A. The responses were caused by the application of ACh to the proximal end-plate zone of a 25-day-denervated muscl e. The upper trace illustrates an initial burst of fibrillatory potentials followed by a decrease in frequency of discharge and amplitude of the potentials. The lower trace depicts the pro­longed contracture in itiated by the ACh. B. The responses were produced by the appl ication of ACh to the proximal end-plate zone of another 25-day-denervated muscle. The upper trace shows the drug-induced abrupt suppression of the fibrillatory potentials; the lower trace denotes the contractu re • In both A and B, the "notch II appearing in the contracture traces does not represent a biphasic response. It is the result of a touch artifact. C. The response was evoked by the appl ication of ACh to an end-plate-free site on a 25-day-denervated muscle. The upper trace illustrates the lack of effect of ACh on the fibrillatory potentials; the lower trace depicts the contracture. A 15 sec 300 mgl ACh 5 X 10-6 pg /pl B 150 pvl III~~~~~~~~~~~~~~~~~: t ACh 5 X 10-6 pg/pl ~ --~- 300 mg I AC hi JI 0 - 5 p 9 / P I 71 Figure 8. The relationship between depolarization and the concentration of ACh. The depolarizations (mY) were measured extracellularly with conventional glass microelectrodes. ACh solution was applied at the tip of the microelectrode by the microdrop technic. Concentrations of ACh are expressed as the log. A. The data were obtained from one site with in a proximal end-plate zone (filled circles) and one end-plate-free area (open circles) of a 20-day­denervated muscle. The volume of each microdrop of drug solution was 1 pl. B. Each response was obtained from a different end-plate-free site of a 21-day-denervated muscle. DTC was administered intravenously into the right femoral vein. The volume of each microdrop of ACh solution was 0.5,.,1.. C. Each response was obtained from a different end-plate-free site of a 20-day-denervated muscle. Atropine was administered intravenously into the right femoral vein. The volume of each microdrop of ACh solution was O.S}JI. A 12 • END - PLA TE ZONE II o END-PLATE-FREE AREA 10 9 > e 8 z 2 7 I-cr N 6 a:: .c..r.. 5 0 Q. 4 ~ Q 3 2 10-6 10-5 10-4 ACh CONCENTRATION (PO Ipl) B 5 ;e: 4 Z :! Ic-r 3 N a:: 0.c.. .r. 2 Q. ~ Q o CONTROL • OTe 10.5mO/kg) 10-5 10-4 10-3 10-2 ACh CONCENTRATION (pg Ipl' C 10 0 CONTROL 9 • ATROPINE (0.511,1 kg) 8 > e 7 z 2 6 I- cr N 5 a:: .c..r.. 4 0 Q. 3 ~ Q 2 10-4 10-3 10-2 ACh CONCENTRATION (PO Ipl , 73 Figure 9. The relationship between ACh-induced depolarization and tension. Depolarization (mV) was measured extracellularly with microelectrodes; ACh solution was applied at the tip of the electrode by the microdrop method. Volume of the microdrops of ACh solution was 0.5 fJl. A. The data were obtained from one site with in a proximal end-plate zone (filled circles) and one end-plate-free area (open circles) of a 20-day-denervated muscle. B. Each set of depolarization and tension responses was obtained from a different end-plate-free site of a 21-day-denervated muscle. DTC was adminis­tered intravenous Iy into the right femoral vein and increased the concentration of ACh required to produce a specific amount of depolarization by approximately one hundred times. For explanation of the numbers adjacent to the open circles, see D below. C. Each set of depolarization and tension responses was obtained from a different end-plate-free site of another 21-day-denervated muscle. Atropine was adm inistered intravenously into the right femora I vein I and increased the concentration required to evoke a given amount of depolarization by approximately one hundred times. D. The responses illustrated are from the experimental data depicted in B. The numbered values on the graph in B correspond to the numbered responses in D. 75 Figure 10. The effects of topically applied ACh on the membrane potential of one fiber and the tens ion of denervated musc Ie. The ACh solution was applied by the microdrop method. Membrane potential changes were measured intracellu larly with conventional glass m icroelectrodes, and the responses were recorded from end-plate-free reg ions of the muscle. A. Responses obtained in a 23-day-denervated muscle. The volume of the microdrop of ACh solution was 0.5 pl. ACh depolarized the membrane (base line) I decreased the amplitude of the fibri Ilatory potentials (vertical spikes), and produced a contracture (lower trace). The action potentials were retouched for photographic clarity. B. Responses obtained in a 22-day-denervated muscle. The volume of the microdrop of ACh solution was 1 fl. ACh depolarized the membrane of the test fiber (upper trace) and evoked a contracture (lower trace); but it did not elicit an action potential from the markedly depolarized muscle cell. A 15 sec 60 mV I 50 mg I t 6 I ACh 5 X 10- pg /p B 15 sec 60 mvl d , .. 1 200 mg I • • ,,,-, b frd I 77 Figure 11. The local nature of the ACh depolarization. The two experimental sets, A and B, are illustrated in the upper portion of the figure. The record i ng el ectrode (tapering, sol id vertical I ine) is plac ed on an end-plate-free region in both sets. The end-plate zones are contained within each pair of broken lines. The position of the microdrop of ACh solution is shown by the closed circles. The volume of the microdrop of ACh solution was 0.5 fl. The effects of ACh are depicted by the traces directly below each experimental set. A. The microdrop of ACh was placed at the tip of the recording electrode, and the resu Iting depolarization was recorded. B. Twenty minutes after the first microdrop was removed from the muscle, a second drop was placed on the same muscle, 3 mm away from the electrode. As illustrated by the trace, under these spatial conditions the electrode did not record the ACh-induced depolarization occurring in the immediate vicinity of the microdrop 0 The trace records mainly background noise. A -:z- II a= " • II ---, t - \\ ~ \, =: I I iii 10mV IOsee ACh 2.5 X 10-5 pg I pi B =!==!!= ! II"., • 2 mV \, \, I I 10 see ACht 2.5 X 10-5 pg I JlI 79 Figure 12. The effects of cholinergic blocking agents on normal neuromuscular transmission of the anterior gracilis muscle. During obturator nerve stimu lation the drugs were adm in istered intravenous Iy into the right femoral vein (at the arrows), and their effects noted on maximal twitch tension. The electrical stimulus was twice that required to produce max­imal twitch tension, and the frequency of stimulation was once every 2 sec. A. The effect of DTC on maximal twitch tension. B. The effect of atropine on maximal twitch tension. C\ .Jill: ....... ~ C\ ...-::: ....... e CI\ e LaJ Z . 0 e... <.,.) l- 0 0 Ct: I- <r: « 81 Figure 13. The effects of catecholamines on the denervated anterior gracilis muscleD Depolarizations were recorded extracellularlywith a glass microelectrode. Drugs were applied by the microdrop method and the volume of the drop was 1 pL Each drop was applied to a different end-plate-free site. Drugs were also administered intra-arterially. Responses in A, B, C, and D were all obtained from the same 24-day-denervated muscle. Responses in E, F, G, and H were obtained from the same 21-day-denervated muscle. Upper trace in each set, depo larization; lower trace, tens ion. A. Topically appl ied norepinephrine produced no effect. B 0 Topically appl ied ACh evoked both depolarization and contracture. Co Intra-arterially administered norepinephrine elicited both depolariza-tion and contracture. Do Intra-arterially administered ACh caused both depolarization and con­tracture. E. Topically applied isoproterenol produced no effect. F. Topically applied ACh evoked both depolarization and contracture. G. Intra-arterially administered isoproterenol produced both depolariza-tion and contracture. H. Intra-arterially admin istered ACh produced both depolarization and contracture. A 'i"07ec lomvl~ __________________ _ 8 ~-------------------- 400mgl __________ _ + NOREPINEPHRINE 2J1g/JlI c D __ "f/II"t::':::...-=_~ ________________ su. ~--------------------- --~----,---------~-: + NOREPINEPHRINE 2 Jig/kg + ACh 50 Jig / kg E F ~--------------------. ~------- t ISOPROTERENOL 5J19/JlI + ACh 7.5XIO-6 J1g / Jl I G H ~--.--- + ~-----~~~~ + ISOPROTERENOL BpQ/kg ACh 75 Jig/kg Name Birthplace Birthdate High School College Un iversity Degrees Fellowships, Awards, and Honors Society Affiliations Publications: VIII. VITA Stuart Allen Turkanis Everett, Massachusetts December 15, 1936 Lynn Engl ish High School Lynn, Massachusetts Massachusetts Coil ege of Pharmacy Boston, Massachusetts 1954-1960 Un i vers i ty of Utah Salt Lake City, Utah 1962-1967 B.S .. in 1958; M.S. in 1960 Massachusetts College of Pharmacy Boston, Massachusetts Scholastic Achievement Award, 1958 Public Health Service Trainee, 1962-1967 Rho Chi Honor Society Master of Science Thesis. Part 1: An evaluation of the comparative analgesic effectiveness of a series of pyrrol idinols and agents with morphine-I ike activity; Part 2: Evaluation of antispasmodic activity in the intact dog. Turkanis, S. A. & Jenkins, H. J. (1963). Evaluation of antispasmodic activity in the intact dog. J. pharm. Sc i. 52, 186-188. Turkanis, S. A., Esplin, D. W., & Zablocka, B. (1964). Evidence for the release­depletion of the inhibitory transmitter by pilocarpine in mice. The Pharma­cologist ~, 192. Williams, J. K., Zablocka, B., Esplin, D. W., & Turkanis, S. A. (1964). Blockade of central inhibition by C-alkylpiperidines. The Pharmacologist 6, 192. Karler, R .. , Turkanis, S. A., & Steinman, S. (l967). Acid precipitation of liver microsomes for the study of drug metabolism. Fedn Proc .. 26, 354. IX. RESEARCH PROPOSALS A. Effects of Atropine on Neuromuscular Transmission in the Anterior Gracilis Muscle of the Rat Turkanis (1967) demonstrated that intravenously administered atropine has a curare-like effect on the anterior gracilis muscle of the rat. Atropine in low doses interrupts neuromuscular transmission in the normal muscle and antagonizes the effects of acetylchol ine (ACh) in the denervated muscle. The purpose of the proposed research is to investigate the character of the atropine blockade of normal neuromuscu lor transm iss ion. The initial step in the investigation is to determine the locus of action of atropine. The effect of atropine on the conduction of the nerve action potential and on the excitability of the muscle will be determined. If neither one of these properties is affected, then it can be assumed that atropine block is due to a junctional site of action. The surmountable nature of the atropine blockad'e of the anterior gracilis muscle suggests that the antagon ism is of the competitive type. If atropine has a mechanism of action similar to that of ~-tubocurarine (DTC), then the character of the neuromuscular blockade produced by atropine must fulfill certain pharma­colog ical cri teria (Za imis, 1959; Koe lie, 1965). The competitive agents raise the threshold of the end-plate to ACh and have the following pharmacolog ical profile. The effects of a competitive blocking agent are antagonized by anti­cholinesterases, potassium, tetanus of the motor nerve, cathodal current applied 85 to the end-plate zones, and a lowering of musc Ie temperature. In addition, the effects of a competitive blocking drug are additive with those of DTC and are enhanced by anodal current applied to the end-plate zones. Unl ike the de­polarizing agents, the competitive neuromuscu lar blocking drugs do not cause transient fasciculations in normal muscle. If the nature of the atropine antagonism meets the criteria described above, then atropine may be appropriately classified as a competitive type of neuromuscular blocking agent in the anterior gracilis muscle of the rat. B. Denervation Contracture Produced by Catecho lam ines It is well known that catecholamines administered parenterally evoke con­tracture in denervated but not in normal skeletal muscle (BlJlbring & Burn, 1936; Luco & Sanchez, 1959; Bowman & Zaimis, 1961). The contracture produced by parenterally administered catecholamines is antagonized by ~-tubocurarine (DTC) and ~-adrenergic blocking agents (Luco & Sanchez, 1959; Bowman & Raper, 1965). Turkanis {1967} demonstrated that topically appl ied catecholamines did not produce contracture but that intra-arterially admin istered catecholam ines produced both depolarization and contracture in the denervated anterior graci I is muscle of the rat. In addition, tachyphylaxis developed to the effects of the catecholamines and cross-tachyphylaxis developed to the effects of parenterally administered ACh. The experimental findings cited above suggest that the catecholamines may produce contracture in the denervated muscle by releasing an active substance in the animal. It is proposed that catecholamines release ACh from an unknown 86 site and that the endogenously released ACh diffuses to the denervated muscle membrane and evokes a contracture. A study of the ability of atropine and DTC to antagonize and physostigmine to enhance the catecholamine-elicited contracture would assist one in deciding upon the validity of such a hypothesis. C. The Trophic Influence of Motor Nerves on Skeletal Muscle There are a number of observations indicating that motor nerves exert an influence, independent of electrical activity, on skeletal muscle. For example, after cutting the motor nerve, the entire surface of the muscle becomes sensitive to the depolarizing effects of ACh (Axelsson & Thesleff, 1959; Mi ledi, 196(0). Upon regeneration of the nerve, the ACh-sensitive area recedes towards the end-plates (Emmelin & Nordenfelt, 1959; Miledi, 1960b). In addition, Luco & Eyzaguirre (1955) demonstrated that the time of onset of denervation phenomena is directly proportional to the length of the distal portion of the severed nerve. Evidence also exists that motor nerves determine the speed of contraction of skeletal muscle (Buller, J. C. Eccles, & R. M. Eccles, 1960). The above-cited resu Its provide support for the concept that motor nerves exert a troph ic influence over skeletal muscle. It has been suggested that motor nerves influence skeletal muscle by extruding a trophic substance(s) from presynaptic terminals (Luco, 1962; Weiss, 1963). At present, the nature of the trophic material is in dispute. Thesleff (1960) studied muscle treated with botulinum toxin and published suggestive evidence that spontaneous emissions of quanta of ACh control the chemosensitivity of skeletal 87 muscle. However, Katz & Miledi (1959), Miledi (1960a, 196Ob, 1963), and Diamond & Miledi (1962) reported direct evidence that ACh is unable to control the chemosensitivity of skeletal muscle. Miledi and his co-workers suggest that ACh does not mediate the trophic influence and that the mediator must be some other, as yet LIn identified substance. In support of such a trophic-transmitter hypothesis are ,the elegant studies indicating proximo-distal convection of substances with in motor nerves 0/Veiss, 1963). As an example of such a study are the resu Its recently reported by Korr, Wi Ik inson, & Chornock (1967). In their experiments rad ioactively labeled com­pounds applied to hypoglossal nuclei in rabbits traveled down the hypoglossal nerves and, after several days, entered the muscle cells of the tongue. Although the motor nerve has the ability to transport substances to the muscle, the existence of a spec ific troph ic transmitter substance has not been demonstrated. The proposed research is designed to obtain direct evidence concerning the existence and nature of a troph ic substance. The following experiments on the denervated frog sartorius, wh ich can be functionally maintained for at least 10 days in a bath at low temperatu res, are suggested. The denervated musc Ie wi II be bathed continuously with extracts of the frog spinal cord, and each day the muscle will be tested with ACh to evaluate the degree of supersensitivity. If the whole extract proves to be effective in decreasing the chemoreceptive area of the muscle, then both the particulate and soluble fractions of the extract will be tested separately. Standard analytical procedures will be employed to study 88 the chemical nature of a potential trophic substance in the extract. In addition, candidate substances selected as a result of the analysis of the extract will be tested on the denervated musc Ie. D. Research Proposal References Axelsson, J. & Thesleff, S. (1959). A study of supersens itivi ty in denervated mammalian skeletal muscle. J. Physiol. 147,178-193. Bowman, W. C. & Raper, C. (1965). The effects of sympathomimetic amines on chronically denervated skeletal muscle. Br. J. Pharmac, Chemother. 27, 313-331. Bowman, W. C. & Zaimis, E. (1961). The action of adrenal ine, noradrenal ine, and isoprenaline on the denervated mammalian muscle. J. Physiol. 158, 24-25P. - BlJIbring, E. & Burn, J. H. (1936). The Sherrington phenomenon. J. Physiol. 86, 61-76. Buller, A. J., Eccles, J. C., & Eccles, R. M. (1960). Differentiation of fast and slow muscles in the cat hind limbs. J. Physiol. 150,399-416. Diamond, J. & Miledi, R. (1962). A study of foetal and new-born rat muscle. J. Phys iol. 162, 393-408. Emmelin, N. & Nordenfelt, I. (1959). Effects of cross-suture and of injected acetylchol ine on supersens itivity of denervated striated musc Ie. Br. J. Pharmac. Chemother. 14, 234-238. Katz, B. & Miledi, R. (1959). Spontaneous subthreshold activity at denervated amphibian end-plates. J. Physiol. 146, 45-46~: Koelle, G. B. (1965). Neuromuscular blocking agents. The pharmacological basis of therapeutics, 3rd Edition, ed. Goodman, L. S. & Gilman, A., pp. 596-613. New York: The Macmillan Company. Korr, I. M., Wilkinson, P. N., & Chornock, F. W. (1967). Axonal delivery of neuroplasmic components to muscle cells. Sc ience, N. Y. 155, 342-345. 89 Luco, J. V. (1962). Physiological studies during degeneration. Acta physiol. latinoam . .!,3., 384-389. Luco, J. Vo & Eyzaguirre, C. (1955). Fibrillation and hypersensitivity to ACh in denervated muscle: effect of length of degenerating nerve fibers. J. Neurophysiol. ~, 65-73. Luco, J. V. & Sanchez, P. (1959). The effect of adrenaline and noradrenaline on denervated skeletal muscle: antagonism be1ween curare and adrenaline- I ike substances. Curare and curare-I ike agents, ed. Bovet, D., Bovet-N itti, F., & Marini-Bettolo, G. B., pp. 405-408. Amsterdam, London, & New York: Elsevier Publishing Company. Miledi, R. (1960a). The acetylcholine sensitivity of frog muscle fibers after complete and partial denervation. J. Physiol. 151, 1-23. Miledi, R. (196Ob). Properties of regenerating neuromuscu lor synapses in the frog. J .. Phys iol. 154, 190-205. Miledi, R. (1963). An influence of nerve not mediated by impulses. Th~ effect of use qnd disuse on neuromuscular functions, ed. Gutmann, E. & Hrftk, P., pp. 35-40. Amsterdam, London, & New York: Elsevier Publishing Company. Thesleff, S. (1960). Supersensitivity of skeletal muscle produced by botu I inurn toxin. J. Physiol. 151, 598-607. Turkanis, S .. A. (1967). Pharmacological and physiological properties of denervated mammal ian skeletal muscle. Ph. D. Thesis, Un iversity of Utah, Salt Lake City, Utah. Weiss, P. (1963). Self-renewal and proximo-distal convection in nerve fibers. The effect of use and disuse on neuromuscu lor ru nctions, ed .. Gutmann, E., & Hn1k, P., pp. 171-183. Amsterdam, London, & New York: Elsevier Pub­lishing Company. Zaimis, E. (1959). Mechanisms of neuromuscular blockade. Curare and curare­like agents, ed. Bovet, D., Bovet-Nitti, F., & Marini-Bettolo, G. B., pp. 191-203. Amsterdam, London, & New York: Elsevier Publishing Company.