Biomedical subjects
Publications and source records attributed to R S Wilkinson.
Nearly 30 years ago, a debate over two disparate models of synaptic vesicle retrieval arose. Subsequent investigations to determine which model was correct proved inconclusive and served only to further the debate. However, recent evidence has begun to suggest that both models are valid and relevant in understanding the complex nature of vesicle recycling.
We have used the activity-dependent probe FM1-43 with electron microscopy (EM) to examine endocytosis at the vertebrate nerve-muscle synapse. Preparations were fixed after very brief neural stimulation at reduced temperature, and internalized FM1-43 was photoconverted into an electron-dense reaction product. To locate the reaction product, we reconstructed computer renderings of individual terminal boutons from serial EM sections. Most of the reaction product was seen in 40-60 nm vesicles. All of the labeled vesicles were clathrin-coated, and 92% of them were located within 300 nm of the plasma membrane, suggesting that they had undergone little processing after retrieval from their endocytic sites. The vesicles (and by inference the sites) were not dispersed randomly near the plane of the membrane but instead were clustered significantly near active zones. Additional reaction product was found within putative macropinosomes; these appeared to form from deep membrane invaginations near active zones. Thus two mechanisms of endocytosis were evident after brief stimulation. Endocytosis near active zones is consistent with the existence of local exo/endocytic cycling pools. This mechanism also might serve to maintain alignment of active zones with postsynaptic folds during periods of activity when vesicular and plasma membranes are interchanged.
To investigate the role of actin in vertebrate nerve terminals, nerve-muscle preparations from garter snake (Thamnophis sirtalis) were treated with the actin-depolymerizing agent latrunculin A. Immunostaining revealed that actin filaments within presynaptic motor terminal boutons were disrupted by the drug. In preparations loaded with the optical probe FM1-43, destaining was reduced by latrunculin treatment, suggesting that transmitter release was partially blocked. Latrunculin treatment did not influence the amplitude or time course of spontaneous miniature endplate potentials (MEPPs). Similarly, endplate potentials (EPPs) evoked at low frequency were comparable in control and latrunculin-treated curarized preparations. Brief tetanic stimulation of the muscle nerve (25 Hz, 90 s) depressed EPP amplitudes in both control and latrunculin-treated preparations. After tetanus, EPPs elicited at 0. 2 Hz in control preparations recovered rapidly (0-5 min) and completely (usually potentiating to above pre-tetanus levels; 130 +/- 11 %, mean +/- s.e.m.). In contrast, EPPs evoked in latrunculin-treated preparations recovered slowly (8-10 min) and incompletely (84 +/- 8 %). The influence of latrunculin on post-tetanic EPPs depended on its concentration in the bath (KD = 3. 1 microM) and on time of incubation. These observations argue that actin filaments facilitate transmitter release rather than impede it. Specifically, actin may facilitate mobilization of vesicles towards the releasable pools.
We have used a sensitive activity-dependent probe, sulforhodamine 101 (SR101), to view endocytic events within snake motor nerve terminals. After very brief neural stimulation at reduced temperature, SR101 is visualized exclusively at punctate sites located just inside the presynaptic membrane of each terminal bouton. The number of sites (approximately 26 sites/bouton) and their location (in register with postsynaptic folds) are similar to the number and location of active zones in snake motor terminals, suggesting a spatial association between exocytosis and endocytosis under these stimulus conditions. With more prolonged stimulation, larger SR101-containing structures appear at the bouton margins. Thus endocytosis occurs initially at distinct sites, which we call "endocytic active zones," whereas further stimulation recruits a second endocytic paradigm.
Dystrophin is a cytoskeletal protein of muscle fibers; its loss in humans leads to Duchenne muscular dystrophy, an inevitably fatal wasting of skeletal and cardiac muscle. mdx mice also lack dystrophin, but are only mildly dystrophic. Utrophin, a homolog of dystrophin, is confined to the postsynaptic membrane at skeletal neuromuscular junctions and has been implicated in synaptic development. However, mice lacking utrophin show only subtle neuromuscular defects. Here, we asked whether the mild phenotypes of the two single mutants reflect compensation between the two proteins. Synaptic development was qualitatively normal in double mutants, but dystrophy was severe and closely resembled that seen in Duchenne. Thus, utrophin attenuates the effects of dystrophin deficiency, and the double mutant may provide a useful model for studies of pathogenesis and therapy.
1. Motor nerve terminals innervating fibres in the transversus abdominis muscle of the garter snake comprise discrete boutons. Using a combination of enzymatic digestion and mechanical manipulation, individual boutons were removed from living terminals for study in isolation. 2. Boutons freed from terminals were usually allowed to remain in their original location on the endplate ('attached' one-bouton synapse). Alternatively, they were removed from the endplate, and then placed on the same or another vacant endplate site to form a 'reconstructed' one-bouton synapse. When removed from the endplate, boutons were 2-4 microns in diameter and nearly spherical in shape, in contrast to the variety of complex shapes seen among boutons still in contact with muscle fibre endplates. 3. Transmitter release was assessed by intracellular recording from the postsynaptic fibre. Boutons produced spontaneous miniature endplate potentials (MEPPs) of nearly normal amplitude; extracellular stimulation elicited endplate potentials (EPPs) which resembled MEPPs. Typical EPP amplitudes fluctuated between zero and five quanta per stimulus. For low-frequency stimulation under normal physiological conditions, mean quantal content, m, averaged 1.4; the binomial number of release sites, n, averaged 2.4; and the binomial probability of release, p, averaged 0.57. Statistics of the quantal fluctuations recorded from single boutons agreed only approximately with predictions of simple binomial theory, the discrepancy being that the apparent number of quanta released exceeded n in 5% of the events. 4. In separate experiments, activity-dependent probes were used to locate rare naturally occurring nerve terminals comprising a single bouton. Activation of these small synapses evoked quantal responses similar to those of attached and reconstructed one-bouton synapses described above.
We have developed a technique, called synaptic reconstruction, that permits nerve terminals of living vertebrate neuromuscular junctions (NMJs) to be isolated and then manually recombined with vacant endplate sites to form functional synapses. By reconstructing NMJs with various combinations of pre- and postsynaptic partners, or with varying degrees of pre- to postsynaptic alignment, the functional properties of the three anatomical components of the NMJ--nerve terminal, endplate, and the alignment between them--may be studied independently. Our experiments thus far indicate, surprisingly, that reconstructed NMJs function nearly normally. Thus, one feature of the intact vertebrate NMJ, precise alignment between presynaptic active zones and postsynaptic secondary folds, is either unnecessary for normal function or, alternatively, is spontaneously reestablished when an isolated terminal and vacant endplate site are placed in contact. We have also utilized synaptic reconstruction to examine a recently described property of NMJs: the regulation of quantal size among motor synapses in one muscle so that larger muscle fibers receive larger single quantal currents. Quantal size appears to be a postsynaptic attribute, suggesting that its regulation is achieved by a postsynaptic mechanism.
1. Postsynaptic responses to spontaneous quantal transmitter release have been compared among neuromuscular junctions in a thin snake muscle. For each junction the type, diameter, and input conductance, G(in) of the postsynaptic muscle fibre were determined. Particularly among fibres of a given type, G(in) was directly correlated with fibre diameter. 2. Miniature endplate potentials (MEPPs) were recorded intracellularly near endplates visualized with Nomarski optics. Mean MEPP amplitude decreased with increasing G(in) among fibres in one muscle. However, the dependence of mean amplitude upon G(in) was not ohmic, as would be expected if the underlying single quantal currents (miniature endplate currents, MEPCs) were of similar amplitude at all junctions. Instead, the relation between MEPPs and G(in) suggested that mean MEPC amplitudes, calculated as mean MEPP amplitude x G(in), increased with increasing G(in). 3. MEPCs were recorded directly using the two-microelectrode voltage clamp technique. Mean MEPC amplitudes depended systematically on G(in), again such that MEPCs were on average larger in fibres with higher G(in). 4. MEPCs were recorded extracellularly from small regions of endplates (underlying a few nerve terminal boutons). Amplitudes of MEPCs depended on G(in) or fibre diameter in the same manner as amplitudes of MEPCs recorded by intracellular voltage clamp. 5. When the anticholinesterase agent neostigmine was added to the bath, amplitude and duration of MEPPs, MEPCs, and extracellular MEPCs increased. However, the systematic dependence of mean MEPC amplitude on G(in) or fibre diameter remained. 6. Evoked subthreshold endplate potentials (EPPs) were recorded under conditions of low extracellular Ca2+. Endplate currents (EPP amplitudes x G(in)) were systematically larger in fibres with larger G(in), indicating regulation of evoked synaptic current in the muscle. The regulation was found to be due to a combination of increased quantal content and larger single quantal currents in larger (higher G(in)) fibres. 7. Synaptic size, assessed either by area of cholinesterase staining or number of terminal boutons, increased with increasing fibre diameter. Assuming that quantal content is proportional to synaptic size, this relation was sufficient to account for the observed increase in quantal content with increasing G(in) among fibres in the muscle, but was not alone sufficient to account for the observed regulation of evoked current. 8. It is concluded that the efficacy of individual transmitter quanta released at the snake neuromuscular junction is regulated such that large muscle fibres receive larger single quantal currents. Regulation of single quantal current contributes substantially to overall regulation of synaptic strength in the muscle.
1. Motor units in the thin transversus abdominis muscle of the garter snake were identified and physiologically characterized in the living state. Motor unit fibres, and fibres chosen randomly to serve as controls, were subsequently excised and subjected to biochemical analyses. 2. The metabolic capacity of fibres was assessed by measuring activities of three enzymes, each representing a different metabolic pathway. The microchemical enzyme assays were performed using enzyme extraction preparations of whole single fibres. 3. Metabolic capacity ranged widely among the muscle's entire fibre population, even among fibres of the same type. In contrast, enzyme activities of twitch fibres belonging to individual motor units were, within analytical error, identical. 4. Twitch contraction times of individual fibres within one motor unit were similar, compared to a wide range of contraction times observed among fibres of the same type but belonging to different motor units. 5. When several motor units were studied in one muscle, a systematic relationship was observed among motor unit tension, enzymatic profile and contraction time. As motor unit tension increased, fibres exhibited greater capacities for glycolytic and high-energy phosphate metabolism, diminished capacity for oxidative metabolism, and faster twitch contraction times. 6. Given the great diversity of metabolic and contractile properties exhibited within the fibre population, the uniformity of such properties within motor units indicates that neural influence dominates over other extrinsic factors present in the microenvironment of the muscle fibres.
1. The transversus abdominis muscle of the garter snake contains fibres of three types: tonic (T), slower twitch (S) and faster twitch (F). Fibre types can be determined by anatomical criteria in living preparations. Individual fibres identified as T, S or F were excised from the muscle and subdivided for two types of biochemical examination. Enzymes of energy metabolism were assayed using quantitative microfluorometric methods. Myosin heavy chain composition was determined by gel electrophoresis. In separate experiments, twitch time-to-peaks of F and S fibres were measured to assess the range of contraction times present within the muscle's twitch fibre population. 2. Metabolic subgroups of fibres were delineated by the relative activities of adenylokinase (AK), lactate dehydrogenase (LDH) and beta-hydroxyacyl-CoA-dehydrogenase (beta OAC). The metabolic subgroups corresponded to the anatomical fibre types. Type F fibres had high levels of enzymes associated with glycolytic (LDH) and high-energy phosphate (AK) metabolism. Type T fibres had high levels of the oxidative enzyme beta OAC. Type S fibres had both types of enzyme activity in intermediate and variable amounts. 3. Three myosin heavy chain isoforms were present in the muscle. Type F and type T fibres each expressed a single isoform, denoted F and T respectively. Type S fibres expressed significant quantities of two isoforms: an isoform unique to this fibre type (denoted S) and the F isoform. 4. Electrophoretic mobility and antibody reactivity of the F myosin heavy chain isoform resembled that of mammalian fast-twitch myosin. By the same criteria, the T isoform resembled mammalian slow-twitch myosin. The S isoform exhibited intermediate characteristics: its antibody reactivity was similar to mammalian fast-twitch myosin, but its electrophoretic mobility was that of mammalian slow-twitch myosin. 5. Based on whole-muscle analysis, two myosin alkali light chains, denoted ALC1 and ALC2, and one myosin regulatory light chain were present. Gel patterns suggested that ALC1 and ALC2 exist as both homodimers and heterodimers. 6. The population of type S fibres within a given muscle exhibited a much wider range of twitch contraction times than did the population of type F fibres. Diversity of contractile properties among type S fibres may result, in part, from differential co-expression of two myosin heavy chain isoforms, together with highly variable ratios of enzymes from two major metabolic pathways. 7. The clear biochemical distinction among fibre types indicates that each type possesses a unique and limited range of physiological properties.(ABSTRACT TRUNCATED AT 400 WORDS)
The distribution of fibre types in the tibialis anterior (TA) muscle of adult mice was examined by means of an immunohistochemical approach, using monoclonal antibodies that recognize different myosin heavy chain isoforms. As has been reported previously, the superficial portion of TA contains almost exclusively type IIB fibres and is almost entirely glycolytic in nature. Following section of the lateral popliteal nerve and rotation of the proximal stump to prevent rematching, it was found that the original pattern was virtually restored within 2 months. One possible explanation for this observation is that the activity pattern of peripheral and deep muscle fibres differs and that this aids in specification of muscle fibre type. Alternatively, the muscle fibres of the superficial portion of TA may be inherently resistant to an alteration of their phenotype with regard to expression of myosin heavy chain.
Fibers of the garter snake transversus abdominis muscle fall into three classes according to contraction speed: faster and slower twitch and tonic. To determine the relationship between these physiologically determined classes and established mammalian fiber types, individual fibers were assayed for key enzymes representing the major energy-generating pathways in vertebrate muscle. Five such enzymes were examined: lactate dehydrogenase, malate dehydrogenase, adenylokinase, fumarate hydratase, and beta-hydroxyacyl-CoA dehydrogenase. The muscle contained three principal metabolic fiber types. Fast-contracting twitch fibers had low-oxidative but high-glycolytic capacity and therefore resembled mammalian-type fast-twitch glycolytic (FG) fibers. Slower twitch fibers were high oxidative-high glycolytic, similar to mammalian-type fast-twitch, oxidative, glycolytic (FOG) fibers. Tonic fibers were high oxidative-low glycolytic; this metabolic profile is characteristic of type slow-twitch oxidative (SO) fibers in mammals. Activity of the enzyme adenylokinase, which in mammals correlates with contraction speed and myosin adenosine triphosphatase (ATPase) activity, separated these reptilian fibers into three groups that are similar but not identical to those delineated by oxidative and glycolytic enzymes. Adenylokinase and beta-hydroxyacyl-CoA dehydrogenase showed the widest range of activities in snake muscle and, therefore, the greatest ability to discriminate fiber types.
We describe a straightforward modification to a conventional fluorescence microscope that permits confocal fluorescence imaging of living and fixed biological material. The modified microscope has been used to examine the morphology of presynaptic terminals at the snake neuromuscular junction. Among our observations is the presence of two discrete compartments within each terminal bouton.
1. The organization of motor units in the single-fibre-thick transversus abdominis muscle of the garter snake has been studied. This small segmental muscle (60-100 fibres) contains three distinct fibre types (faster twitch, F; slower twitch, S; and tonic, T) which are predominantly arranged in the repeating pattern F, T, S, T, F, T, etc. 2. Motor-unit maps were obtained by activating an individual motor axon and identifying all of the muscle fibres innervated by that axon, using either the activity-induced uptake of extracellular marker molecules to label presynaptic terminals of the stimulated axon, or systematic intracellular recording to identify muscle fibres activated by the axon. 3. Each muscle contained three types of motor units (F, T and S) that corresponded to the three types of fibres. All of the muscle fibres in a motor unit were of the same type. Each segmental muscle contained approximately the same number of motor units: one to two faster twitch, three to four slower twitch, and three tonic. 4. Each motor unit was dispersed widely throughout the muscle. Fibres within a motor unit were neither clustered nor anticlustered. This suggests that despite the fact that axons are constrained to innervate fibres of the appropriate type, the distribution of each motor unit does not generate the alternating pattern of fibre types. 5. In several experiments, all of the twitch motor units in one segmental component of the muscle were mapped. The projection of any one axon appeared random not only with respect to the muscle's fibre type pattern, but also with respect to the innervation supplied by other axons. 6. Twitch motor units were arranged according to a hierarchy of sizes. In each muscle examined, the largest motor unit was faster twitch; a single faster twitch motor axon usually innervated all of the faster twitch fibres in the muscle (fourteen to twenty-four fibres). This was followed by three to four slower twitch motor units which varied in size from eight to ten fibres to very small motor units containing only four to five fibres. 7. Each tonic motor axon innervated an average of ninety-three end-plates per segmental muscle. The relatively large size of tonic motor units compared to twitch motor units is related to the ability of tonic muscle fibres to retain polyneuronal innervation into adulthood, both by providing five to seven end-plate sites per fibre, and by allowing terminal boutons from different tonic motor axons to co-innervate the same end-plate.(ABSTRACT TRUNCATED AT 400 WORDS)
The snake transversus abdominis muscle is an extremely simple segmentally repeating muscle containing 80 to 100 muscle fibers in a single-fiber-thick sheet. This muscle exhibits a striking pattern of muscle fiber types: twitch fibers alternate with tonic fibers and, among the twitch fibers, slower and faster contracting subtypes also alternate. Thus, in many regions of the muscle the pattern of fiber types is: faster twitch, tonic, slower twitch, tonic, faster twitch, tonic, and so on. The existence of a spatial pattern of fiber types, perhaps discernible in this muscle because of the muscle's extreme geometrical simplicity, provides good evidence for an intrinsic component to muscle fiber differentiation.
Three studies were conducted to investigate the endocrine and ovulatory responses of the prepubertal gilt to exogenous estradiol and gonadotropins. In Study One, prepubertal gilts of 190 days of age were injected s.c. with pregnant mare's serum gonadotropin (PMSG) or physiological saline (SAL). Following PMSG injection, circulating levels of estradiol-17 beta (E2) increased. This increase was followed by a surge of luteinizing hormone (LH), estrus, a rise in progesterone (P4) levels, and ovulation. None of the gilts given SAL had increased levels of E2, LH or P4, and none ovulated. In Study Two, prepubertal gilts of 165 days of age were treated with varying doses of PMSG. A positive correlation was observed between dose of PMSG and peak levels of E2 (r = 0.83, P less than 0.001) and between dose of PMSG and number of corpora lutea (r = 0.96, P less than 0.001). In Study Three, gilts were treated at ages of 70 to 190 days with estradiol benzoate (EB), PMSG, or corn oil plus saline (CO/SAL) followed in 72 to 96 h by human chorionic gonadotropin (hCG) or SAL. All gilts treated with EB at 100 to 175 days of age had two surges of LH at an approximately 24-h interval. Gilts responding to EB at 70 and 190 days had only one surge of LH. Gilts of 100 days of age or older responded to PMSG with a single surge or two surges of LH. Ovulation in response to treatment was observed in gilts of 100 days of age or greater but not at 70 days. The conclusions drawn from these studies are that 1) PMSG-induced ovulation is preceded by an increase in circulating levels of E2 and in some gilts by a surge of LH, and 2) prepubertal gilts are able to respond to exogenous endocrine stimulation with either a single surge or multiple surges of LH at 70 to 190 days but are unable to ovulate in response to exogenous gonadotropins until 100 days of age.
1. Receptor potential and tension have been recorded from isolated Golgi tendon organs in response to sinusoidal stretch. Responses depended on amplitude and frequency of stretch and on the initial (resting) tension of the preparation. 2. Both tension and receptor potential behaved as power functions of stretch amplitude over most of the range corresponding to physiological tendon strains. However, for very small stretch amplitudes (less than 8 microns), a more linear response was seen. Those characteristics of responses that depended on stretch amplitude behaved similarly at all frequencies examined. 3. Frequency dependence of tension was slight. Its character, a gradual monotonic increase in response with increasing stretch frequency and a constant phase lead of a few degrees, did not change over the examined frequency range from 0.12 to 80 Hz. In contrast, receptor potential displayed a marked frequency dependence, increasing rapidly with increasing frequency of stretch in the range from approximately 1 to 20 Hz, then slowly declining as frequency was further increased. 4. Changes in initial tension of the preparation produced marked parallel changes in the amplitude dependence of tension and receptor potential. Frequency response was not significantly affected. 5. By comparing tension and receptor potential responses, the relative contributions of mechanical and electrical properties of the receptor to the sensory transduction process was examined. The present results suggest that in tendon organs the observed nonlinear dependence on amplitude of stretch originates primarily in the mechanical stage of transduction. Dynamic sensitivity, however, seems largely attributable to ionic processes within the sensory terminal membranes.
In isolated cat muscle spindles the receptor potential responses of primary and secondary endings as well as tension responses to sinusoidal length changes in the steady state have been analysed. 1. At a given stimulus frequency, receptor potential per unit length change (receptor potential gain) in both primary and secondary endings is constant when displacement is less than about 10 micrometer. With larger stretches, receptor potential gain decreases approximately as a power function of displacement, the gain of primary endings decreasing more rapidly with increasing displacement than that of secondary endings. Tension per unit length change (tension gain) shows a similar constant range above which it also decreases as a power function of displacement. 2. In spite of the large reduction in gain at high displacement amplitudes, response wave forms remained essentially sinusoidal. The gain reduction results principally from a displacement-dependent non-linearity which has a rapid onset and slow decay. 3. Receptor potential and tension responses to small amplitude sinusoidal stretch depend, in a parallel manner, on the initial length of the preparation. 4. Both receptor potential and tension responses are highly dependent on frequency of sinusoidal stretch. In primary endings receptor potential gain increased as a power function of frequency over the range 0 . 01 to about 40 Hz, above which frequency the gain decreased; phase advance remained relatively constant up to 10 Hz then decreased to become a phase lag at higher frequency. In secondary endings receptor potential gain remained fairly constant between 0 . 01 and 1 Hz then rose as a power function of frequency but less steeply than in primary endings. 3. The possible mechanisms underlying these findings are discussed.