Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

Ultrastructure of the phasic stretch receptor in the crayfish abdominal nerve cord

Summary The ultrastructure of the abdominal ganglionic cord stretch receptor of the crayfishPacifastacus leniusculus is described. This bilaterally-paired, segmentally-repeating phasic receptor monitors stretch applied to the central nervous system itself. It consists of a connective tissue mass closely applied to the medial margin of each medial giant fibre, into which ramifies a collection of specialized terminal dendrites originating from branches (primary dendrites) of a single axon. The connective tissue consists of an electron-opaque matrix in which are embedded many short, electron-lucent, tubular structures whose lumens are continuous with the matrix. Some filamentous material penetrates the connective tissue from its boundaries, and glial cells are present. The primary dendrites are irregular in size and orientation, and contain many microtubules and much filamentous material. The terminal dendrites are of consistent diameter and longitudinal orientation, containing very regularly-spaced microtubules with no microfilaments. The terminal dendrites contain a well-defined cytoskeletal tube or lamina 6 nm thick, evenly spaced about 25 nm below the plasma membrane and connected to it by filamentous material 5 nm in diameter, which is deposited in rings or helices. This lamina arises just at the point where the primary dendrites gave rise to the terminal dendrites. Its function is not known, but it shows some similarities to the subaxolemmal lamina found in some regions of spike initiation.     

Dose-response curve of glutamate applied by superfusion to crayfish muscle synapses

Summary Single muscle fibers were space clamped to a membrane potential of –75 to –80 mV, and the synaptic currents elicited byl-glutamate (gEPSCs) were recorded. The bathing solutions flowing across the fibers at high speed could be switched rapidly and repeatedly through valves actuated by solenoids. Glutamate solutions were applied for periods of 7 s or 1 s, and the responses to repeated applications were averaged. For glutamate concentrations of 10–50 mol/l, applied for 7 s, the gEPSCs reached a steady state. In this concentration range the amplitude of the gEPSC rose steeply proportional to the powern=2.5 ton=6 (average of 12 experimentsn=4.0) of the glutamate concentration. At higher concentrations, after rising for a few seconds the gEPSC was reduced by desensitization. At 500 mol/l glutamate complete desensitization was reached with an approximate time constant of less than 1 s. The glutamate concentration that elicited a half maximum gEPSC wasK=70 mol/l. If glutamate was superfused only for 1 s, similar dose-response curves were observed. In these experimentsn was between 4 and 6. The results obtained by superfusion agree quantitatively with those published for electrophoretic applications.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Structure of membranes in crayfish muscle: comparison of phasic and tonic fibres

Summary Membranes of two crayfish muscles with different contraction speeds were studied with freeze-fracture replicas and thin sections. A fast-contracting, short sarcomere phasic muscle, the tail flexor, and a slowly contracting long sarcomere tonic muscle, the carpopodite flexor, were chosen for this study. Membranes examined included the plasmalemma, clefts, T-system, Z-tubules and sarcoplasmic reticulum (SR).We found distinct differences in the distribution of these membranes: T-system and clefts are more elaborate in the tail flexor, while SR is more extensive in the leg flexor. The tail flexor apparently lacks Z-tubules. These differences were more obvious in freeze-fracture replicas than in thin sections.In freeze-fracture replicas, both junctional and non-junctional T-tubule membranes can be distinguished from Z-tubules by content of intramembranous particles. The junctional regions of T-system and surface membranes contain large (10–11 nm) intramembranous particles that are absent from non-junctional parts of these membranes. There is also a class of particles on the junctional SR fracture faces that differs from intramembranous particles on non-junctional SR. These junctional specializations are similar in long and short sarcomere fibres.     

The action of inhibitory drugs on nerve terminals in crayfish muscle

Summary The presynaptic inhibitory effects of -amino-butyric acid (GABA) and related drugs on the excitatory and inhibitory terminal potentials were studied using extracellular microelectrodes.Triphasic conducted action potentials recorded from the excitatory nerve fiber in the vicinity of the terminal were altered to monophasic positive potential changes after application of inhibitory drugs. These drugs thus can block conduction near to the terminal.Diphasic potential changes recorded near to the excitatory terminal (e.n.t.p.s) were made monophasic positive after application of inhibitory drugs. Neural presynaptic inhibition had the same effect. Also during drug induced inhibition therefore the point of block of conduction in the excitatory terminal shifted centrally.The potentials recorded from the inhibitory nerve terminal were not or very little affected by applied GABA.GABA in concentrations that blocked excitatory transmission did not change the frequency of spontaneous excitatory potentials.It is concluded that presynaptic inhibition by GABA and related drugs mimicks in all respects known neural presynaptic inhibition. The probable mechanism of this inhibition is a conductance increase without much potential change in the excitatory nerve terminal.

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Zusammenfassung Die hemmenden Effekte von GABA und verwandten Drogen auf die Potentiale der erregenden und hemmenden Nervenendigungen wurden mit Hilfe von extracellulären Mikroelektroden-Ableitungen untersucht.Von erregenden Nervenfasern in der Nähe der Endigung wurden triphasische Aktionspotentiale abgeleitet. Nach der Gabe von hemmenden Drogen wurden die Potentiale monophasisch positiv; diese Substanzen könnten also einen Block der Fortleitung des Aktionspotentials herbeiführen.Nahe der erregenden Endigungen abgeleitete diphasische Potentiale (e.n.t.p.) wurden nach Applikation von hemmenden Drogen kleiner und monophasisch positiv. Präsynaptische Hemmung durch Reiz des hemmenden Nerven hatte dieselbe Wirkung. Es verschiebt sich also auch während der Hemmung durch Drogen die Stelle, an der die Fortleitung des Aktionspotentials aufhört, zentralwärts.Von der hemmenden Nervenendigung abgeleitete Potentialänderungen wurden durch GABA nicht oder sehr wenig beeinflußt.GABA in Konzentrationen, bei denen die erregende synaptische Übertragung unterbrochen war, veränderte nicht die Frequenz der spontanen erregenden Potentiale.Die Befunde zeigen, daß die präsynaptische Hemmung durch GABA und verwandte Substanzen von der präsynaptischen Hemmung über den hemmenden Nerven nicht unterschieden werden kann. Wahrscheinlich ist der Mechanismus dieser Hemmung eine Erhöhung der Leitfähigkeit der Zellmembran der erregenden Nervenendigung, ohne daß dabei das Membranpotential sich wesentlich verschiebt.

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With 5 Figures in the Text

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This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.     

Unloaded shortening after a quick release of a contracting,single fibre from crayfish slow muscle

Summary The time course of shortening at zero load was studied by the slack test method during tetanic contractions in isolated, single, slow muscle fibres of the crayfish. In 28 of 32 shortenings (from 14 different fibres) a biphasic shortening was seen, which consisted of an initial high-velocity phase lasting 3.3–20.8 ms and a following slow-velocity phase lasting for the entire time examined (up to 89.2 ms). Provided that the shortening occurred uniformly along the fibre length, velocity in the initial phase, V₁, of the biphasic shortening was 14.4±3.4 (mean±SD,n=10) m s^–1 per half sarcomere at Lo, the slack length, at 20°C, while that in the second phase, V₂, was 7.4±1.4 m s^–1 per half sarcomere. Lowering temperature decreased both V₁ and V₂ with Q₁₀=1.4 for V₁ and 2.0 for V₂. Lowering the external Ca concentration from 15 mM, the standard, to 2 mM resulted in a tetanic tension below one-third of that at 15 mM Ca and decreased both V₁ (t test; p<0.01) and V₂ (p<0.001). Prestretching the fibre to 1.5 Lo had no significant effect on V₂ (p<0.3) but increased V₁ (p<0.001). The distance shortened during the initial high-velocity phase, LV₁, was 4.0±1.8% Lo (mean±D,n=10) at 20°C or about 0.14 m per half sarcomere on average. LV₁ was independent of the tetanic tension level when it was changed by lowering the external Ca concentration or temperature in the same fibre. Prestretching the fibre to 1.5 Lo, at which the sum of the active and the resting tension was lower than Po at Lo in two of three fibres, increased LV₁ significantly (p<0.001). The independency of LV₁ from the tension level indicates that the initial high-velocity phase was not from shortening of some inert components in the fibre. One possibility is that the initial high-velocity phase was brought about by an acceleration of shortening by a compressive force, the origin of which has been discussed. The slow-velocity phase seemed to result from the crossbridge turnover with little exogeneous stress on myofilaments. Four different fibres exhibited an unloaded shortening with a constant velocity during the entire time examined (29.9–61.8 ms). This type of shortening had a velocity between the usual V₁ and V₂ values, suggesting that a compressive force accelerated the shortening during the entire time.     

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.     

Effect of lithium on veratridine-induced quantal and non-quantal release from inhibitory nerve terminals in crayfish muscle

Muscle fibres of small crayfish were voltage clamped and superfused for about 10 min with Li⁺ saline (Na⁺ replaced by Li⁺) which contained 5 mmol/l glutamate to desensitize excitatory postsynaptic receptors. Then 100 mol/l veratridine were added to the superfusate which caused strong asynchronous quantal release of inhibitory transmitter. However, in the presence of Li⁺ strong inhibitory quantal release was only transient. It could be activated a second time by removal of Li⁺ and readministration of Na⁺. From the total of 0.7 to 1.1 million quanta released by veratridine only about 30–35% could be released in Li⁺ saline. The voltage clamp DC-currents recorded during veratridine-induced quantal release suggested that a nonquantal release component is additionally involved. This non-quantal release component was most prominent during the period of quantal release in Li⁺ superfusate while it was less obvious during the second enhancement of quantal release in normal saline. Together with previous results (Martin and Finger 1988) it may be concluded that quantal release, but not non-quantal release, is decreased by Li⁺ in the nerve terminals.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Economic force maintenance in a phasic smooth muscle of mollusca

The time course of the ability of active shortening was compared to that of isometric contractile force during contraction-relaxation cycles of the pedal retractor muscle of Mytilus by measuring the maximal shortening velocity and the rate of force redevelopment after a quick release. The contractile force had its peak at 2 s after stimulus initiation and decayed with a half relaxation time of 8.3 s at 10 degrees C. At 6 s after stimulus initiation, both the maximal velocity of shortening and the rate of force redevelopment had decreased to 30% or less of the initial values, while 70% of peak force was retained. The results suggest a mechanism of economic force maintenance with a reduced rate of crossbridge cycling.     

Postembryonic development of rectifying electrical synapses in crayfish: physiology

Summary In a previous paper we showed that the ultrastructure of the giant fibre to motor giant synapse of crayfish changes in the first few weeks after hatching from having predominantly the appearance of a chemical synapse to having the appearance of an electrical synapse. This is parallelled by a behavioural change from non-giant fibre-mediated to giant fibre-mediated tailflips. In this paper we describe the physiology of the giant fibre to motor giant synapse over this period. We find the following: (1) The giant fibre to motor giant synapse usually transmits spikes 11 from the day of hatching. (2) The synapse operates by electrical transmission from the day of hatching, when no connexons are apparent at the ultrastructural level. (3) The synapse has no detectable chemical component, even at an age when the predominant type of junctional apposition has the ultrastructural appearance of a chemical synapse. (4) Inhibitory chemical synapses occur onto the motor giant at the day of hatching, and these show similar physiological characteristics to those which occur onto the motor giant in adults. (5) In some preparations, the giant fibre to motor giant electrical synapse shows rectification similar to that in the adult, but in most cases both depolarizing and hyperpolarizing current injected into the medial giant spreads to the motor giant. (6) Current spread from the medial giant to the motor giant is increased by hyperpolarizing the motor giant neuron, even when medial giant to motor giant transmission is apparently non-rectifying. (7) Both the giant fibre and the motor giant have resting potentials of about –90 mV. There is no standing difference in resting potential as there is in the adult. This may explain the apparent lack of medial giant to motor giant rectification observed in most preparations.     

Tonic depolarization of excitatory nerve terminals in crayfish muscle by high concentrations of extracellular potassium

At voltage-clamped fibres of the claw opener muscle of small crayfish, spontaneous quantal release of excitatory transmitter elicited by raising extracellular K+ to 100 mM was investigated. On application of the high K+ concentration, the rates of quantal release increased to n = 10,000-25,000 quanta/s within 10 s, and thereafter declined exponentially, either with a single (tau congruent to 15-40 s) or with two (tau 1 congruent to 15-40 s, tau 2 greater than 70 s) time constants. The total number of quanta released per trial ranged from s = 200,000 to 800,000 quanta. The results were derived by means of the fluctuation analysis technique.     

The distribution of intracellular acetylcholine receptors and nuclei in avian slow muscle fibres during establishment of distributed synapses

Summary The distribution of intracellular acetylcholine receptor was studied by¹²⁵I--bungarotoxin autoradiography as a measure of the local acetylcholine receptor synthesis at junctional and extrajunctional sites in single fibres of the developing anterior latissimus dorsi muscle of the chicken. Large (longer than 2 m) acetylcholine receptor clusters characteristic of synaptic contacts were localized by immunofluorescence with anti-acetylcholine receptor antibodies. The distance between acetylcholine receptor clusters at embryonic day 11 was 166 ± 10.5 m and this distance did not increase despite growth until after 4 days posthatch. The distance between acetylcholine receptor clusters subsequently increased proportionately with the increase in the length of fibres. Intracellular acetylcholine receptors were labelled with¹²⁵I--BGT after first blocking cell-surface acetylcholine receptor with unlabelled -BGT, and treatment with saponin. Intracellular acetylcholine receptor represented about 5–15% of total cellular acetylcholine receptor. Cycloheximide experiments indicated that 80–90% of intracellular acetylcholine receptor examined represented newly synthesized acetylcholine receptor. The spatial distribution of this pool, studied by autoradiography, was determined in relation to the acetylcholine receptor clusters labelled with anti-acetylcholine receptor antibody. Between embryonic day 11 and posthatch day 14 there was a continual increase in intracellular acetylcholine receptor at both junctional and extrajunctional parts of the fibres, but with the greater increases occurring at the junctional regions. Peaks of intracellular acetylcholine receptor became associated with an increasing number of acetylcholine receptor clusters so that by posthatch day 14 there was an 80% correspondence. The accumulation of newly synthesized intracellular acetylcholine receptor under acetylcholine receptor clusters was not the result of the aggregation of nuclei at these sites, suggesting that a higher rate of acetylcholine receptor synthesis per nucleus develops at distributed synaptic sites on anterior latissimus dorsi fibres.     

Calcium dependence of quantal release triggered by graded depolarization pulses to nerve terminals on crayfish and frog muscle

Quantal transmitter release was measured in small portions of neuromuscular junctions by means of a perfused macro-patch-clamp electrode. Release was elicited by graded current pulses through the recording electrode (excitation blocked by TTX). On increasing the stimulation current from a threshold amplitude, release rose steeply for several orders of magnitude and finally approached a saturation level of about 10 quanta/pulse. Reduction of the Ca concentration in the perfusate of the electrode, Ca_e, depressed the saturation level of release relatively little and had practically no effect on the threshold current amplitude, as long as the Ca concentration in the superfusion of the bath, Ca_b, remained high. When Ca_b was reduced too, the depression of release was more severe. The dependence of release on Ca_e was determined for a large range of Ca_e for saturating depolarization pulses. In crayfish, at 0 Ca_b, in double-logarithmic release-Ca_e plots the maximum slope was on average 3.9, and this slope dropped to on average 2.1 in 13.5 mM Ca_b. In frog, at 0 Ca_b, the respective double-logarithmic slope was 3.5, while in 1.8 mM Ca_b this slope declined dramatically, the rate of release decreasing on average only by a factor of 3.8 from 10 mM to 0.02 mM Ca_e. These results are interpreted by the assumption that the resting Ca concentration in the terminal, Ca_ir, has strong influence on the rate of release due to depolarization pulses in low Ca_e, and that Ca_b has control on Ca_ir in the terminal.Supported by the Deutsche Forschungsgemeinschaft     

Anti-GABA antibodies label a subpopulation of chemical synapses which modulate an electrical synapse in crayfish

Summary Antibodies raised against gamma-aminobutyric acid (GABA) were used to stain sections from the crayfish abdominal nervous system, and the sections were examined under the electron microscope using a protein-A/gold conjugate secondary label. Sections were taken through the third ganglionic root, and through the interganglionic connective at the base of the third root posterior to the ganglia. The third root contains two very large motor axons, a non-GABAergic excitor (Motor Giant; MoG), and a GABAergic inhibitor (Flexor Inhibitor; FI). Only one of the two large axons stained positively for GABA, confirming that the antibody has high specificity for GABAergic neurones.The MoG is driven by powerful electrical synapses from the giant fibres, but also receives inhibitory chemical synaptic input which can gate the excitatory input. There is no physiological evidence for any other form of chemical input. However, at the ultrastructural level, the MoG is postsynaptic to three types of chemical profiles; SE-type containing round agranular vesicles, SI-type containing pleomorphic vesicles, and SM-type containing a mixture of round agranular and dense-cored vesicles. There is a highly differentiated staining pattern of these three synaptic types. Only the SI-type profiles stain positively with the GABA antibody, while the SE- and SM-type do not show significant staining. This suggests that the MoG can under some circumstances receive chemical input other than GABAergic inhibitory input. These other types of input have yet to be physiologically identified.     

Regeneration and remyelination of Xenopus tadpole optic nerve fibres following transection or crush

Optic nerves of stage 54-56 Xenopus laevis tadpoles were either transected or crushed, and subsequent Wallerian degeneration, regeneration, and remyelination were examined. After 4 days, normal myelinated fibres were no longer present in the distal stump, and only a few unmyelinated fibres remained. After 10-13 days, the distal nerve consisted mainly of a core of reactive astrocytes with enlarged processes and scattered oligodendrocytes which persisted throughout the degenerative period. Regenerating axons traversed the site of the lesion and extended into the distal stump within 13-15 days. As regeneration progressed, astrocytic processes extended radially from the optic nerve's central cellular core and formed longitudinal compartments for regenerating axons. Between 15-19 days, a few regenerating fibres were remyelinated and by 35 days, more axons were surrounded either by thin collars of oligodendrocyte cytoplasm or by 1-3 spiral turns of myelin membrane. By 95 days, the number of myelinated fibres had increased to about 50% of those present in control nerves. Their myelin sheaths were normal in appearance and thickness relative to their respective axon diameters. The largest axons were surrounded by compact sheaths with 4-9 lamellae.