Changes in spontaneous and evoked release of transmitter induced by the crotoxin complex and its component phospholipase A2 at the frog neuromuscular junction.

The time course and characteristics of the changes induced by the action of the crotoxin complex and of its component phospholipase A₂ on transmission at the frog neuromuscular junction was studiedin vitro at single endplates using intracellular recording techniques. Both the crotoxin complex and phospholipase A₂ induced variable changes in spontaneous transmitter release in which bursts of miniature endplate potentials alternated with periods when the frequency of miniature endplate potentials was stable but their amplitudes were markedly heterogeneous. The bursts of miniature endplate potentials were of sudden onset and of a high initial frequency, and their duration and incidence were highly variable both at one endplate and between endplates. These changes in the frequency and amplitude of miniature potentials occurred both before and after the abolition of neurally evoked endplate potentials and in the presence of tetrodotoxin. At a late stage in crotoxin intoxication, potassium depolarization of the motor nerve terminals failed to increase spontaneous transmitter release. Both the crotoxin complex and phospholipase A₂ reduced the amplitude of endplate potentials although phospholipase A₂ was less effective. The course of depression of the quantal content of evoked transmitter release induced by the crotoxin complex was occasionally interrupted by a brief facilitation of response; these irregularities in the rate of depression were more clearly seen when endplate potentials were recorded at partially curarized endplates exposed to either the crotoxin complex or to phospholipase A₂. At the stage of abolition of endplate potentials an increase in the external concentration of Ca^{2 +} enabled the evoked response to be restored temporarily. The crotoxin complex and phospholipase A₂ had no effect on the input resistance of the sarcolemmal membrane, but raised the threshold stimulus required to initiate an action potential at concentrations which affected transmitter release. Crotapotin did not affect any of the measured parameters even at high concentrations.The diversity of changes observed in the properties of spontaneous and evoked transmitter release, suggests that the crotoxin complex and phospholipase A₂ may induce two kinds of alterations in the structure of the presynaptic membrane as a result of the action of phospholipase A₂, viz (a) the formation of transient instabilities with an increased Ca^{2 +} inflow and (b) prolonged disturbance of the calcium channel and/or the synchronized release of subunits at the active zone.     

A comparison of miniature end-plate potentials at normal,denervated, and long-term botulinum toxin type A poisoned frog neuromuscular junctions

The effects of denervation and long-term botulinum toxin type A (BoTx) poisoning on miniature endplate potentials (m.e.p.p.s) in the frog were studied with intracellular microelectrode recording. BoTx reduced the frequency of m.e.p.p.s. to less than 1% of the level seen in untreated frogs, leaving a large percentage of tiny m.e.p.p.s and slow-rising m.e.p.p.s (slow m.e.p.p.s). Unlike what is observed in the rat, the frequency of slow m.e.p.p.s never increased above the low rate measured in the untreated controls, and in fact slightly but significantly decreased after BoTx. A comparison of the m.e.p.p.s seen after BoTx poisoning (BoTx m.e.p.p.s) and m.e.p.p.s seen after denervation (Schwann m.e.p.p.s) revealed many similarities between the two including amplitude and time-to-peak distributions, temperature Q₁₀ values and responses to several drugs and procedures. However, it was concluded that BoTx m.e.p.p.s do not originate from the Schwann cells because 1. denervation of BoTx-paralysed frogs abolishes all m.e.p.p.s and 2. the drug 4-aminoquinoline affects BoTx m.e.p.p.s and Schwann m.e.p.p.s in opposite ways, increasing the frequency of the former while almost eliminating the latter. BoTx m.e.p.p.s and Schwann m.e.p.p.s probably represent similar processes of secretion which are non-specific in nature, having a lower energy barrier than for normal release and not originating from specialized areas of transmitter release.     

Spontaneous synchronization by 4-aminopyridine of acetylcholine release in the neuromuscular junction

The effect of 4-aminopyridine (4AP) on the character of acetylcholine release was investigated by intracellular recording of spontaneous synaptic activity in an isolated rate phrenic nervediaphragm preparation. No significant changes were found in the mean frequency and amplitude of miniature end-plate potentials (MEPP) in response to 4AP in concentrations of 1·10^–6 to 1·10^–3M. Meanwhile, 4AP caused the appearance of large spontaneous EPP capable of inducing spreading action potentials. 4AP changed the character of distribution of MEPP amplitudes, converting it to polymodal; in some cases the principal mode was shifted into the region of lower values. It is concluded that 4AP changes the character of acetylcholine release and potentiates spontaneous synchronization.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Department of Pharmacology, Faculty of Internal Medicine and Hygiene, I. M. Sechenov First Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 259–262, September, 1979.     

The extent of sprouting of remaining motor units in partly denervated immature and adult rat soleus muscle

The soleus muscle has been partially denervated by cutting its principal nerve in newborn and mature rats of the inbred strain AO. These rats commonly have an abnormal (aberrant) second nerve supplying the soleus muscle. After various survival times the sizes of the remaining motor units were determined. In adult rats the remaining motor units sprouted to about four times their average normal size. In contrast, the initially larger motor units of partially denervated newborn muscles became smaller in size and remained significantly smaller than motor units which had sprouted in partially denervated mature muscles.We conclude that the elimination of the neonatal polyneuronal innervation takes place even in partially denervated muscle. A substantial number of the muscle fibres are consequently left without innervation. In the adult rat an average increase of about four times appears to be the maximum extent of sprouting of remaining motor units in partially denervated rat soleus.     

Choline acetyltransferase in transected nerves, denervated muscles and Schwann cells of the frog: correlation of biochemical electron microscopical and electrophysiological observations.

The activity of choline acetyltransferase was increased in the peripheral nerve stumps of transected sciatic nerves of frogs (Rana esculenta) 5–21 days after the transection but had fallen markedly by the beginning of the second month. The increase of choline acetyltransferase activity began at a time when large myelinated fibres were still unaffected by degeneration according to both ultrastructural and electrophysiological criteria, whereas the small fibres were undergoing degeneration. It preceded the appearance of the miniature end-plate potentials attributable to the Schwann cells in the denervated sartorius muscle, which occurred 22–25 days after nerve section. The steep fall of choline acetyltransferase activity in the degenerating nerve coincided with the final stages of destruction of the large myelinated axons. One-fifth of control choline acetyltransferase activity was found in the degenerated nerve even 3 months after nerve section, when all axonal remnants were completely resorbed and the peripheral nerve stump was almost exclusively composed of proliferated Schwann cells and of the Büngner bands, formed by their processes. The activity of choline acetyltransferase in denervated sartorius muscle diminished, starting from the fifth day after nerve section. It did not fall below 60% of control level even 3 months after denervation, but it could not be excluded that in the muscle some of the synthesis of acetylcholine observed during assays of choline acetyltransferase activity was due to non-specific acetylcholine-synthesizing activity of carnitine acetyltransferase.The observations strongly suggest that choline acetyltransferase is present in the Schwann cells of degenerated frog nerves and that the enzyme is newly synthesized, rather than taken up from degenerating axons. The synthesis of choline acetyltransferase in degenerating nerves is not restricted to the Schwann cells in direct contact with the muscle fibres; apparently, it depends on the relationship between the Schwann cells and axons, and not solely (if at all) on the relationship between the Schwann cells and muscle fibres.     

Nicotinic acetylcholine receptor-ion channels involved in synaptic currents in bullfrog sympathetic ganglion cells and effects of atropine

The nicotinic acetylcholine receptor-ion channels (AChR channels) of the bullfrog sympathetic ganglion cells were studied with a two-electrode voltage clamp technique. The decay phase of the fast excitatory postsynaptic current (fast e.p.s.c.) in B-type neurones followed a double exponential function whose time constants were 3.2 and 8.0 ms at –60 mV and increased with membrane hyperpolarization. Likewise, the decay phase of the fast e.p.s.c. in C-type neurones was double-exponential with time constants of 4.4 and 12.3 ms (at –60 mV). The miniature e.p.s.c. in B-type neurones also decayed with a double exponential function (2.7 and 15.4 ms at –100 mV). Analysis of acetylcholine-induced current fluctuations revealed the power spectral density distribution of a double Lorentzian function which yielded the time constants of elementary events [_noise(f) and _noise(s): 1.7 and 29.7 ms, respectively, at –100 mV] and the averaged elementary conductance (: 7.8 pS).The amplitude of fast e.p.s.c. and the time constant of the fast component of its decay phase decreased during the initial (acute) phase (within 15 min) of the action of atropine (3 M), but recovered during the later (chronic) phase (more than 30 min after application) of the action. The slow component was affected by atropine in a manner similar to the fast component during the acute phase.During the chronic phase, however, the slow time constant recovered and exceeded the control value. Furthermore, this prolongation remained for at least 1 h after the removal of atropine. _noise(s) during the chronic phase of the action was also prolonged to 50.3 ms, while [_noise(f) (2.1 ms) was similar to the control value. The amplitude and quantal size of the fast excitatory postsynaptic potential were also decreased during the acute phase of atropine action, and recovered during the chronic phase. Interestingly, they were reduced transiently during the course of removal of atropine from the bath.These results revealed that the decay phases of the fast e.p.s.c. and miniature e.p.s.c. have two components which are explained either by the existence of two types of AChR channels having different open times or by a single type having three states with rate constants of certain relationships in the bullfrog sympathetic ganglion cells and suggested that their open forms are blocked by atropine only transiently and later desensitized to the blocking action.     

The effect of acetylcholine on the function and structure of the developing mammalian neuromuscular junction

Isolated soleus muscles from rats aged 9–12 days were exposed to acetylcholine for 2 h in normal Krebs solution. This treatment caused changes in the ultrastructural appearance of the neuro-muscular junction and a significant reduction of axon profiles per endplate. Nevertheless, most neuro-muscular junctions remained functional, since the ratio of the indirectly to directly elicited contraction was not reduced.If muscles were exposed to acetylcholine in Krebs solutions containing 12m M Ca²⁺ instead of the normal 1.9 m M, the ultrastructural changes produced by acetylcholine were more severe, and the number of axon terminals per endplate was further reduced so that many endplates became completely denervated. This was also reflected in the impaired function of the nerve-muscle preparation; the ratio of the indirectly to directly elicited contraction decreased and about 40% of the muscles fibres became functionally denervated. Addition of curare to the incubating medium prevented the functional deterioration of the preparation.Addition of the protease inhibitors leupeptin and pepstatin protected the nerve terminals from the damaging effects of acetylcholine in Krebs solution containing 12m M Ca²⁺ and the number of axon profiles per endplate remained normal. The functional deterioration was also much reduced when protease inhibitors were included in the incubation medium.These results suggest that acetylcholine causes the activation and release of proteolytic enzymes in developing muscles. The response is mediated by calcium and may have a role in the removal of superfluous nerve-muscle contacts during development.     

Potentiation of inhibition by general anaesthetics in neurones of the olfactory cortex in vitro

Pentobarbitone, phenobarbitone, methohexitone, chloralose and alphaxalone produced 10-fold increases in the duration of an inhibitory post-synaptic conductance (i.p.s.c.) as recorded intracellularly from neurones of the guinea-pig olfactory cortex in vitro. Higher concentrations slightly depolarised these neurones and reduced their input resistances (R_i), presumably a spontaneous activation of the inhibitory conductance. The excitatory potentials were also depressed. Ketamine, halothane and urethane doubled the i.p.s.c. duration. Higher concentrations depressed synaptic activity and the action potential, as did lignocaine. Ketamine also increased R_i. These results confirm the idea that these compounds produced anaesthesia by prolonging inhibition (accompanied by a depression of the e.p.s.p. with some anaesthetics).     

The facilitating effect of gangliosides on the electrogenic (Na+/K+) pump and on the resistance of the membrane potential to hypoxia in neuromuscular preparation

The effects have been investigated of a mixture of gangliosides from beef brain cortex (GM₁, GD_1a, GD_1b and GT₁) either added to the bathing medium or injected intraperitoneally on muscle fibres and nerve terminals in mouse diaphragm. The electrogenic (Na⁺/K⁺) pump activity of muscle fibres enriched with sodium was increased by 38% after 2-h pretreatment with gangliosides (5×10^–8 mol ·l^–1). Muscles from animals treated with gangliosides did not show the substantial depolarization of the resting membrane potential (RMP) in K⁺-free solution (6 h) shown by control muscles. Further, treatment with gangliosides slowed the changes in muscle fibre RMP and frequency of the miniature end-plate potentials in oxygen deprived muscles.     

Effects of pindolol,sotalol and the optical isomers of propranolol on muscle action potentials and depolarization-secretion coupling in the rat

Muscle action potentials and miniature end-plate potential frequency were studied in different concentrations of pindolol, d-and l-propranolol and sotalol using intracellular microelectrode recording from rat phrenic nerve-diaphragm preparations, d-and l-propranolol at concentrations of 10 to 40 mg/1 and pindolol at 20 to 100 mg/1 slowed down the rate of rise, prolonged rise and half-decay times and reduced the overshoot of the muscle action potentials. Sotalol had similar effects but only at higher concentrations (100 to 200 mg/1). The drugs had no effect on the increase in the miniature end-plate potential frequency obtained with depolarization by increased extracellular potassium concentration.     

Further investigations on the effect of denervation and pH on the conductance change at the neuromuscular junction of the frog

Summary Currents induced by acetylcholine application at the voltage-clamped frog end-plate, were measured over a large range of membrane potentials. Due to a non-linearity of the current-voltage curve, the directly-measured reversal potential may be quite different from the value classically determined by extrapolation (linear regression) of the measurements made at potentials below spike threshold.Denervation and changes of external pH were found to alter the shape of the current-voltage relation, but not the directly-measured reversal potential. These effects are tentatively explained on the basis of changes in the ratio: time-to-peak for [ACh] reaching the receptors/mean life-time of the open synaptic channels. Possible changes in cooperativity are also considered.This work was supported in part by Délégation Générale à la Recherche Scientifique et Technique, A.C.C. Membranes Biologiques, aide No 74.7.0191     

Influences of morphology and topography of motoneurons and muscle spindle afferents on amplitude of single fiber excitatory postsynaptic potentials in cat

Summary Excitatory postsynaptic potentials (e.p.s. p.s) elicited by impulses in single muscle spindle afferent fibers from the medial gastrocnemius (m.g.) muscle were recorded intracellularly from homonymous and heteronymous motoneurons in order to study factors that influence the amplitudes of such responses. Impulses in large afferent fibers elicited larger single-fiber e.p.s.p.s than those in smaller afferents. Mean e.p.s.p. amplitudes were related exponentially to afferent conduction velocities of both Ia and spindle group II fibers. The closer a motoneuron was to the spinal entry point of an afferent fiber, the larger was the mean e.p.s.p. amplitude evoked in it. Impulses in the same afferent fiber elicited larger e.p.s.p.s in small than large motoneurons when the two cells were located at the same craniocaudal levels. Other factors being equal, the single-fiber e.p.s.p.s evoked in homonymous and heteronymous motoneurons were approximately equal in amplitude. Relatively simple morphological and topographical explanations for the findings were advanced and their functional significance for orderly recruitment in partitioned and unpartitioned reflexes was described.     

Summation of fibre potentials and the e.m.g.-force relationship during the voluntary movement of a forearm

A linear mathematical model of the electromyogram (e.m.g.) has been developed for the biceps muscle. The number of motor units (and therefore muscle fibres) contributing to the resultant e.m.g. at any stage of movement has been found from the force analysis of elbow flexion. The depths of various motor units and the phase difference between the recruitment of any two motor units have been formulated using a spiral spread of recruitment sequence. The attenuation of individual motor-unit action potentials due to varying depths has been taken into consideration, and due regard has been taken of the length-tension diagram of a muscle while performing the force analysis. Attention has been focused on the flexion of the elbow joint, in which a method of finding the individual contribution of the biceps and brachialis muscles has been developed and applied. The results predicted by the model have been verified by experiments. The model can also be extended to the e.m.g. of other fast skeletal muscles. The conditions and limitations for such generalisations have been stated and discussed.     

Effect of electrode parameters on the bandwidth of the surface e.m.g. power-density spectrum

The effect of the electrode configuration (unipolar against bipolar), the electrode separation, the electrode position and the electrode contact area on the bandwidth of the power density spectrum of the surface e.m.g. has been studied. The dependence of the half-power point bandwidthB on the interelectrode distanced can be described by the regression function B_Hz=810/d_mm+58. The unipolar electrode configuration yields an e.m.g. signal with the smallest bandwidth. Neither a variation of the electrode position nor a variation of the electrode contact area has a significant effect on the bandwidth of the e.m.g.     

The fine structure of the ependymal surface of the recessus infundibularis in the rat

Summary The surface of the recessus infundibularis of the third ventricle has been studied with the scanning and transmission technique in normal and experimental material.Surface specializations such as microvilli, craters and areas of discontinuous lining are described. Supraependymal cells and fibres have been found; some of these cells form wide-meshed networks. The supraependymal fibres may be regular or varicose; the former seem to perforate the ependyma.With the transmission electron microscope the supraependymal cells are divided into three categories: nerve cells, lymphocytes and dense cells. Two fibre populations are distinguished: thin profiles (nerve fibres) and thick profiles (nerve terminals). Axosomatic and axoaxonic synapses are described.Synapses between supraependymal fibres and ependyma cells have also been found.     

The effects of calcium and magnesium on statistical release parameters at the crayfish neuromuscular junction

1. Transmitter release at excitatory neuromuscular junctions of crayfish muscle was studied at low temperature by recording synaptic potentials with extracellular micro-electrodes.2. Increasing the Ca concentration in the bathing solution produced an increase in the average number of quanta released per nerve stimulus (m). Increasing the Mg concentration resulted in a decrease in m.3. Statistical analysis of fluctuations in the quantal release from trial to trial, assuming binomial statistics, indicated that both the changes in m were due to changes in the average quantal release probability (p).     

The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian neuromuscular junction

1. Electron micrographs of nerve terminals in rat phrenic nerve—diaphragm preparations have been studied. This has been done before and after prolonged nerve stimulation. The effectiveness of nerve stimulation has been monitored by intracellular micro-electrode recordings from the muscle cells.

2. Characteristic changes in the form and distribution of the nerve terminal mitochondria were noted after nerve stimulation.

3. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were significantly greater in tissue taken 2 and 3 min after nerve stimulation, than in unstimulated preparations.

4. The long and short diameters of the synaptic vesicle profiles less than 1800 Å from the synaptic cleft were measured. Analysis of the distribution of the diameters indicated synaptic vesicles to be basically spherical structures. Estimates of synaptic vesicle volume were made from the measurements. Synaptic vesicle volume was significantly reduced in tissue taken 2 and 4 min following nerve stimulation.

5. If hemicholinium, a compound which inhibits acetylcholine synthesis, was present during the period of nerve stimulation, much greater reductions in synaptic vesicle volume occurred. Synaptic vesicle numbers in the region of nerve terminal less than 1800 Å from the synaptic cleft were also reduced, compared with unstimulated control preparations.

6. These results are regarded as support for the hypothesis that the synaptic vesicles in nerve terminals at the mammalian neuromuscular junction represent stores of the transmitter substance, acetylcholine.

     

Corticosteroids and neuromuscular transmission: Electrophysiological investigation of the effects of prednisolone on normal and anticholinesterase-treated neuromuscular junction

The effect of prednisolone on indirectly stimulated rat muscle twitch was investigated at normal and prostigmine-treated neuromuscular junctions. In vivo, predenisolone up to 150 mg/kg body weight did not affect twitch contraction in normal animals. In neostigmine-pretreated animals, however, doses between 12.5 and 90 mg/kg could entirely abolish the anticholinesterase-induced twitch augmentation. In vitro, prednisolone produced a depressant effect on the twitch of a normal phrenic nerve diaphragm preparation which could amount to 20%. When the preparation was pretreated with neostigmine the augmented twitch could be depressed by 10^–3 to 10^–6 mol/l prednisolone to levels below the untreated control. Part of this effect is owing to a suppression of the neostigmine-induced, stimulus-bound repetitive firing of the motor nerve terminals, but to explain the full effect a further inhibitory action on neuromuscular transmission must be assumed. The latter could be accounted for by a depolarizing interaction of prednisolone and neostigmine on the nerve terminals resulting in conduction block. An action of prednisolone on postsynaptic receptors could also be considered. Such effects of the glucocorticoid might contribute to the exacerbation of muscular weakness occasionally observed in patients with myasthenia gravis at the beginning of steroid therapy.     

The effects of pH on the conductance change evoked by iontophoresis in the frog neuromuscular junction

Summary The amplitude of the electrophoretically evoked end-plate potential increases with changing the pH of the bathing solution from 9.4 to 5.4 at room temperature. This change is not observed at lower temperature. The underlying current (e.p.c. _I ) is slightly decreasing at room temperature by lowering the pH. The relationship between the amplitude of the e.p.c. _I and membrane potential is highly non-linear at pH 9.4, while it is quite linear at pH 5.4. The time course of the e.p.c. _I is changed neither by different pH, nor by different membrane potential. The data suggest that during the e.p.c. _I , the mediator (ACh), the receptor (R) and the mediator-receptor complex are in equilibrium: the amplitude of the e.p.c. _I will thus depend on the affinity constant of the reversible reaction between ACh and R. It is concluded that by decreasing the pH, the affinity constant is decreased.