Development of Drosophila larval neuromuscular junctions: maintaining synaptic strength

In spite of the available information about the development of Drosophila neuromuscular junctions, the correlation between nerve terminal morphology and maintenance of synaptic strength has still not been systematically addressed throughout larval development. We characterized the growth of the abdominal longitudinal muscle 6 (m6) and the motor terminals Ib and Is that innervate it within segment 4. In addition, we measured the evoked excitatory junction potential (EJP) amplitudes while the Ib and Is axons were selectively recruited. Regression analysis with natural log transformation of response variables indicated that the developmental curves for m6 and the motor axons Ib and Is were best fitted as second order polynomial regressions during larval development. Initially Is terminals are longer and possess more synaptic varicosities at the first instar stage. The Is terminals also grow faster in subsequent developmental stages. The growth of nerve terminals and their target m6 are not proportional although tightly correlated. This results in a larger average muscle area innervated by a single varicosity as the animal develops. The amplitudes of the EJPs of Ib and Is neurons show no developmental difference in their amplitudes from the first to the late third larval instar. The Is axon consistently produced larger EJPs than the Ib axon at each developmental stage. The time constants for both rising and decay phases of EJPs increase exponentially throughout larval development.The results presented not only help in quantifying the normal development of Drosophila neuromuscular junctions, but also provide a framework for future investigations to properly interpret developmental abnormalities that may occur in various mutants.     

Serotonin modulates neuromuscular transmission in frogs

The effect of 5-hydroxytryptamine (serotonin) on neuromuscular transmission in frog skeletal muscle was studied using voltage clamp technique. Serotonin produced no effect on end-plate currents during low frequency electrical stimulation of the motor nerve, but increased the amplitude depression of multiquantal currents during high-frequency stimulation similar to motor commands fired by motoneurons. It was shown that the inhibitory effect of serotonin on neuromuscular transmission is determined by slow potential-dependent block of open ionic channels in the postsynaptic membrane accumulating during rhythmic activation of the synapse.     

Heterogeneity in synaptic transmission along a Drosophila larval motor axon

At the Drosophila melanogaster larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-100 boutons onto the muscle it innervates. How transmission strength is distributed among the boutons of the NMJ is unknown. To address this, we created synapcam, a version of the Ca2+ reporter Cameleon. Synapcam localizes to the postsynaptic terminal and selectively reports Ca2+ influx through glutamate receptors (GluRs) with single-impulse and single-bouton resolution. GluR-based Ca2+ signals were uniform within a given connection (that is, a given bouton/postsynaptic terminal pair) but differed considerably among connections of an NMJ. A steep gradient of transmission strength was observed along axonal branches, from weak proximal connections to strong distal ones. Presynaptic imaging showed a matching axonal gradient, with higher Ca2+ influx and exocytosis at distal boutons. The results suggest that transmission strength is mainly determined presynaptically at the level of individual boutons, possibly by one or more factors existing in a gradient.     

Possible role of thiamine in neuromuscular transmission

Pyrithiamine (50 mg/kg), a thiamine antagonist, decreased the muscle twitches of the rat masseter muscle at stimulation frequencies above 1 Hz 40–80 min after an i.v. injection. The post-tetanic potentiation (PTP) induced by nerve stimulation of the masseter muscle was abolished by pyrithiamine. Administration of thiamine restored the muscle twitches at stimulation frequencies above 1 Hz and the PTP. The muscle twitches elicited by direct muscle stimulation were not affected by pyrithiamine treatment. The abolishment of the PTP was accompanied by a decrease in thiamine and thiamine-diphosphate. The pyruvate level in the blood was unchanged after pyrithiamine treatment. Oxythiamine, on the other hand, had no effect on the PTP but increased the pyruvate level in the blood. Fern extract which contains thiaminase I also abolished the PTP–an effect reversible by the addition of thiamine. The frequency-induced depression of the muscle twitches induced by pyrithiamine was similar to the effect of low doses of d-tubocurarine (8 μg/kg). The results support the hypothesis that thiamine may play a role in neuromuscular transmission.     

Properties of the larval neuromuscular junction in Drosophila melanogaster.

The anatomy and physiology of the Drosophila larval neuromuscular junction were studied. 2. The dependence of muscle resting potentials on [K+]o and [Na+]o follows the Goldman-Hodgkin-Katz equation (PNa/PK=0-23). Chloride ions distribute passively across the membrane. 3. The mean specific membrane resistance of muscle fibres is 4-3 X 10(3) omega cm2, and the mean specific membrane capacitance is 7-1 muF/cm2. The muscle fibre is virtually isopotential. 4.Transmitter release is quantal. Both the miniature excitatory junctional potential and the evoked release follow the Poisson distribution. 5. Transmitter release depends on approximately the fourth power of [Ca2+]o. If Sr2+ replaces Ca2+, it depends on approximately the fourth power of [Sr2+]o. Mg2+ reduces transmitter release without altering the fourth power dependence on [Ca2+]o.     

The effects of hypoxia on neuromuscular transmission in a mammalian preparation

1. The rat diaphragm-phrenic nerve preparation in vitro failed to contract in response to nerve impulses after 10-20 min exposure to solutions containing 95% N(2) and 5% CO(2) (hypoxic solutions) at temperatures between 33 and 38 degrees C. Intracellular recording revealed that end-plate potential (e.p.p.) amplitudes fell below the firing threshold for muscle fibres and then disappeared probably because of block of intramuscular nerve conduction.2. In curarized and Mg-paralysed preparations the reduction in e.p.p. amplitudes was found to be due to a fall in their quantal content. In about half of the Mg-paralysed preparations, however, and in curarized preparations after repeated exposures, there were increases in quantal content of e.p.p.s during hypoxia.3. Miniature end-plate potential (m.e.p.p. frequency increased in a cyclic fashion during hypoxia and this increase was largely suppressed in the presence of a raised extracellular Mg concentration. M.e.p.p. amplitude increased (range 0-100% of control value) after about 20 min hypoxia.4. Post-tetanic potentiation of e.p.p. amplitudes and m.e.p.p. frequency was reduced after exposure to hypoxic solutions. During hypoxia the e.p.p. amplitude potentiation was reduced but the m.e.p.p. frequency potentiation was augmented.5. There was an increase in the post-synaptic sensitivity to carbamylcholine after 20 min hypoxia which was sufficient to explain the increase in m.e.p.p. amplitude. Other post-synaptic changes were a fall in membrane potential (average 6 mV after 20 min) and a fall in membrane resistance after 30-60 min exposure to hypoxia.6. The effects of hypoxia upon neuromuscular transmission were partially explained by reduction of active transport of sodium and potassium ions and consequent depolarization of nerve and muscle.     

Digoxin facilitates neuromuscular transmission in mouse diaphragm

Low concentration of digoxin (3 nM) facilitated spontaneous and evoked release of neurotransmitter acetylcholine thereby increasing the frequency of miniature end-plate potentials, amplitude of single end-plate potentials, their quantum content and the plateau level in the bursts during stimulation of the phrenic nerve at rates of 4, 7, and 50 Hz. These effects were prevented by blockade of ryanodine receptors with ryanodine (10–20 μM). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 10, pp. 364–368, October, 2007     

The margin of safety of neuromuscular transmission

1. The margin of safety for neuromuscular transmission in the tibialis and sartorius muscles of the cat has been determined by measuring the ratio by which end-plate depolarization produced by succinylcholine, decamethonium, octamethonium or iodocholine is antagonized, in the presence of neuromuscular block produced by tubocurarine, gallamine or DF-596. The estimate of the margin of safety was independent of the particular drugs chosen for the measurement.2. To produce threshold block to indirect stimulation once every 10 sec, a fractional occupancy by the antagonist of 0.76 +/- 0.05 (S.D.) was required; for nearly complete block, an occupancy of 0.917 +/- 0.16 (S.D.) was required. These figures correspond to factors of safety of 4.1 and 12 for the most sensitive and the most resistant groups of fibres respectively.3. The interaction between the agonists and the antagonists, when tested over a wide range of dosage, did not conform with the conditions of full competitive equilibrium. It was concluded that this arose, not because of some interfering non-competitive process, but because, during the relatively brief exposure to agonist, the equilibrium between the antagonist and the receptors is not significantly disturbed. An analysis of this condition of quasi-equilibrium is given. A correction downwards of the direct estimates of the margin of safety is required, but this proves to be small, about 8%, and may not be significant.4. The safety factor diminished when the motor nerve had been cut more than 5 hr; it is suggested that this represents an early sign of nerve degeneration.5. With dog sartorius muscle, results similar to those in the cat were obtained. But for deep block in the rabbit, the safety factor was only about 4.6. The existence of a substantial margin of safety influences considerably the interpretation of the time course of action of blocking drugs, and of comparisons between responses to nervous excitation and drug injection.     

The effect of procaine on neuromuscular transmission

1. The mechanism of procaine action on post-synaptic receptors for acetylcholine was studied by recording the end-plate current at membrane potentials ranging from about +30 to about -140 mV.

2. It has been found that at resting membrane potential of about -60 to -80 mV the end-plate current has a fast initial and a slow late component. During hyperpolarization of the muscle fibre the amplitude of the slow component is depressed and its half-time lengthened. When the membrane potential is inverted the difference in the time course of both components is much less pronounced or absent.

3. It is suggested that procaine modifies the receptor response induced by acetylcholine, and that this modification is dependent on membrane potential.

     

Action of tetanus toxin on neuromuscular transmission

Summary Comparison of the effects produced by tetanus toxin and proserine (neostigmine) demonstrated in experiments on cats that tetanus toxin was not a cholinesterase inhibitor. There exists a functional antagonism between the action of the tetanus toxin and that of proserine. At the same time, functional synergism was found to exist between the action of the toxin and that of d-paracurarine as evidenced by the fact that curare block developed more rapidly in the toxin-poisoned muscle than in the normal one. With increase of intoxication periods there was a drop in the action potential amplitude in the muscle, and curare block developed more rapidly. A characteristic response of the intoxicated muscle to indirect rhythmic stimulation with a fairly high frequency (pessimal for the normal muscle) was an increase in the action potential during the initial stimuli, and a relative maintenance of its value during the later stimuli.The results of this work permit a conclusion that tetanus toxin disturbs the neuromuscular transmission. The possible mechanisms of this effect are discussed. It is very likely that tetanus toxin influences the excretion of the mediator by the presynaptic apparatus.Presented by V. V. Parin, Active Member, Academy of Medical Sciences, USSR Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 58, No. 10, pp. 65–70, October, 1964