Glutamatergic motoneurons in the stomatogastric ganglion of the mantis shrimp Squilla oratoria

1. Transmitters of motoneurons in the stomatogastric ganglion (STG) of Squilla were identified by analyzing the excitatory neuromuscular properties of muscles in the posterior cardiac plate (pcp) and pyloric regions. 2. Bath and iontophoretic applications of glutamate produce depolarizations in these muscles. The pharmacological experiments and desensitization of the junctional receptors elucidate the glutamatergic nature of the excitatory junctional potentials (EJPs) evoked in the constrictor and dilator muscles. The reversal potentials for the excitatory junctional current (EJC) and for the glutamate-induced current are almost the same. 3. Some types of dilator muscle show sensitivity to both glutamate and acetylcholine (ACh) exogenously applied. The pharmacological evidence and desensitization of the junctional receptors indicate the glutamatergic nature of neuromuscular junctions in these dually sensitive muscles. The reversal potentials for the EJC and for the ACh-induced current are not identical. 4. Glutamate is a candidate as an excitatory neuro-transmitter at the neuromuscular junctions which the STG motoneurons named PCP, PY, PD, LA and VC make with the identified muscles. Kainic and quisqualic acids which act on glutamate receptors are potent excitants of these muscles. Extrajunctional receptors to ACh are present in two types of the muscle innervated by LA and VC. 5. Neurotransmitters used by the STG motoneurons of stomatopods are compared to those of decapods.     

Neurotransmitters of motor neurons in the stomatogastric ganglion of an isopod crustacean, Ligia exotica

Neurotransmitters of motor neurons in the foregut muscles of an isopod Ligia exotica were identified by recording changes in membrane potential to exogenously applied glutamate and acetylcholine. The effects of antagonists, tubocurare and joro spider toxin, on excitatory junctional potentials evoked by nerve stimulation and by iontophoretic application of glutamate and acetylcholine provided additional evidence for identification. The junctional receptors were desensitized by putative neurotransmitters. Glutamate is a candidate as an excitatory neurotransmitter at the neuromuscular junctions in intrinsic muscles of the gastric mill and pylorus, and acetylcholine is a candidate in the extrinsic muscles of the gastric mill and cardiopyloric valve.     

Ultra rapid calcium events in electrically stimulated frog nerve terminals.

Fast calcium events occurring in cytoplasmic organelles after a single electrical stimulus were investigated by electron spectroscopic imaging (an electron microscope technique that reveals total calcium with high sensitivity and spatial resolution) in quick frozen presynaptic terminals of the frog neuromuscular junction. In resting preparations synaptic vesicles showed a prominent calcium signal whereas mitochondria were mostly negative and only some of the cisternae of the endoplasmic reticulum were clearly positive. In preparations quick frozen 10 ms after the application to the nerve of a single, supramaximal electric stimulus, no obvious change was observed in synaptic vesicles, while calcium levels rose to high values in the endoplasmic reticulum cisternae and in the matrix of mitochondria. Voltage-induced influx of Ca(2+) within synaptic terminals appears therefore to induce an extremely rapid uptake into selected organelles. The possible physiological role of this response is discussed.     

Regulation of tetanus toxin-inhibited transmitter secretion in the neuromuscular junction in a calcium-free medium

The effect of Ca2+ removal from the external medium on regulation of the release of the synaptic transmitter in the tetanus toxin (TT)-inhibited neuromuscular junctions was studied on a rat phrenicodiaphragmal preparation with the aid of the conventional microelectrode technique of recording synaptic activity. As the external concentration of calcium was decreased from 2 to 0 mM, the frequency of miniature end plate potentials remained unchanged in the preparations isolated 3 to 3.5 h after intramuscular injection of TT (10(5) MLD for mouse). TT considerably reduced activation of the transmitter release, caused in intact synapses by ouabain (0.1 mM) and repetitive stimulation of the diaphragmatic nerve (50 imp/s). The data obtained indicate that in the TT-inhibited motor nerve terminals, the level of the transmitter release does not depend on the external concentration of calcium and that TT damages some of the intracellular sources of calcium.     

The frog neuromuscular junction revisited after quick-freezing-freeze-drying: ultrastructure, immunogold labelling and high resolution calcium mapping

Until now, most ultrastructural studies on the neuromuscular junction have been carried out on samples first exposed to chemical treatments--with fixatives and/or dehydration agents--that are known to induce, or to be inadequate to prevent, artefactual changes of the native state. We report here on the potential of a physical approach to the preparation of samples that combines quick-freezing and freeze-drying (with or without exposure to OsO4 vapours) followed by direct embedding of the samples in various resins. Thin sections from physically processed frog neuromuscular junctions, when compared to their chemically fixed counterparts, exhibit an overall excellent preservation, with the organelles retaining their native density and shape. These preparations were also investigated by electron spectroscopic imaging and electron energy loss spectroscopy, obtaining high resolution maps of native total calcium distribution within the nerve terminal. Finally, thin sections from analogously processed, however unfixed, preparations embedded in Lowicryl, were immunogold labelled before exposure to OsO4. Nerve-muscle preparations treated this way exhibited adequate preservation of ultrastructure and revealed the distribution of synaptophysin with high sensitivity and resolution. In conclusion, we provide an overview of the potential of the quick-freezing-freeze-drying approach in the study of the neuromuscular junction function.     

Reinnervation of adult muscle in organ culture restores tetrodotoxin sensitivity in the absence of electrical activity.

Strips of denervated adult mouse diaphragm muscle maintained in organ culture were reinnervated by nerve processes growing out from explants of embryonic mouse spinal cord. In vivo, following denervation, the action potential loses its sensitivity to tetrodotoxin; this sensitivity is regained upon reinnervation. Similarly, action potentials in cultured muscle fibres were insensitive to tetrodotoxin, and sensitivity was restored in muscle fibres that became reinnervated in vitro. Tetrodotoxin sensitivity was also restored in cultured muscle fibres reinnervated in the continuous presence of d-tubocurarine, but it was not induced by 4 days of direct electrical stimulation of noninnervated muscles. We conclude that developing nerve terminals can exert a trophic action on adult muscle fibres that is independent of electrical activity in the muscle.     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

Effect of cytostatics on the development of denervation disorders in skeletal muscles

Experiments on rats were made to study the effect of cytostatics on the rest membrane potentials (RMP) of muscle fibres and chemosensitivity of the botulinum toxin (BT) poisoned m. soleus. Intramuscular injection of the sublethal dose of BT on the 5th day evoked the blockade of the synaptic neuromuscular transmission, depolarization of the muscle cells and the decreased sensitivity to acetylcholine. Daily intraperitoneal injections of vincristine (25 micrograms/100 g) and fluorouracil (5 mg/100 g) to rats did not affect the development of the neuromuscular transmission blockade induced by BT. The cytostatics did not change the RMP of the myocytes or chemosensitivity of the normal muscles. However, both the drugs prevented the depolarization of myocytes and the decreased chemosensitivity of the muscles paralyzed with BT. It is assumed that the delayed appearance of the cytostatic-induced denervation is a consequence of the suppressed division of the satellite cells.     

Glutamate uptake by a stimulated insect nerve muscle preparation

Recent reports suggest that glutamate may be the excitatory neuromuscular transmitter in insects. In this study, glutamate uptake by isolated cockroach nerve muscle preparations was investigated by means of chemical and electron microscope radioautographic techniques. We found that the preparation had a high affinity for glutamate and that nerve stimulation enhanced glutamate uptake. Chemical studies showed that the average tissue concentration of glutamate bound during a 1 hr incubation period in 10^-5 M glutamate-³H after nerve stimulation was 2.8 x 10^-5 M. Less than 1% of the radioactivity was present in the perchloric acid-precipitated protein fraction. Using electron microscope radioautography, we observed that sheath cells showed the highest glutamate concentration of all cellular compartments. Uptake was greater at neuromuscular junctions than in other regions of the tissue. The data suggest a possible mechanism for transmitter inactivation and protection of synapses from high blood glutamate.     

Nerve-muscle networks in the accessory gland tubules of a male insect: Structural and physiological properties

The accessory gland tubules of the cockroach Leucophaea maderae, (F.) are innervated by the phallic nerves which arise from the terminal ganglion. Tubule preparations stained with methylene blue showed that a fine network of nerve fibres covers the entire surface of the tubule with putative nerve cells at various points. Muscle fibres of the tubules were arranged in an irregular lattice and appeared as flattened elipses approx 8–10 μm across and 1.5 μm in thickness. These muscles were monomyofibrillar with a poorly developed T-tubule system and a diffuse Z band. Innervation of the muscles showed abundant evidence of neurosecretomotor-type terminals. Clear evidence was also obtained for a multiterminal-type innervation. Accessory gland tubules had a basic wave-like pattern of motion in vitro which showed a wide variation in the time-course of each swing (1–50 s). A twisting and curling motion of the tubules was also recorded. Spontaneous junctional potentials were observed in accessory gland preparations isolated from the terminal ganglion. The duration for junction potential ranged between 30–140 ms. Action potentials recorded from muscles had a slower rise time and a longer duration (230–530 ms).     

Temporal changes in activity during destruction of the thoracic ventral eclosion muscle of the tsetse fly.

The spontaneous intracellular activity of the thoracic ventral longitudinal eclosion muscle (VLEM) of Glossina is described for the period from eclosion up to a short time before the final breakdown of recorded fibres. The VLEM comprises a single motor unit with no inhibitory input. The firing frequency of the motor unit declines over 5 h after eclosion and leg release. Over a period of inactivity lasting between 19-24 h in the sample fibres, there is no loss of resting membrane potential and occasional miniature potentials. The inactivity is ended by the sudden onset of biphasic potentials very different in form to the motor potentials and having a greatly reduced amplitude. These potentials fired at 6 Hz. lasted 2-4 h and ended with a rise in frequency to 25 Hz. No further activity is recorded and the fibres are observed to lose their striations and rigor. Experiments to characterize the ionic basis of activity in the VLEM have been done on spontaneous and evoked activity. Like other insect muscles, the VLEM has a major Ca2+ potential but unlike insect skeletal muscles, it also appears to have a TTX-sensitive component. This Na+ component is revealed by pretreating the system in Na(+)-free-choline saline, or by treatment with TEA in Ca(2+)-free saline. Neither EGTA nor cobalt abolish this potential. Addition of EGTA does not inhibit nerve evoked activity suggesting that the VLEM neuromuscular junction is in some way protected. The similarity of this muscle to insect visceral muscles is discussed.     

Effect of uncoupling agents of oxidative phosphorylation on the spontaneous release of transmitter from insect motor nerve terminals

1. The effect of uncoupling agents of oxidative phosphorylation, potassium warfarin and carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (p-CCP), on the spontaneous release of transmitter was studied at the neuromuscular junction of cockroach muscles. 2. The agents produced a large increase in the frequency of occurrence of miniature excitatory postsynaptic potentials (MEPSPs). This increase also was observed in calcium-free saline. 3. The results may be explained on the hypothesis that the increase in the spontaneous release is due to the increase in free calcium concentration derived from an intracellular origin in the terminal. The mitochondria may play an important role in regulating the intracellular calcium concentration in the nerve terminals of insect muscles.     

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP_8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.     

Supersensitivity to ACh in muscles after prolonged nerve block.

Sciatic nerves of rats and tibial nerves of rabbits were kept anaesthetized in situ for periods of 3-11 days by applying silastic cuffs containing lidocaine base or marcaine hydrochloride. To insure a more uniform release of the rapidly diffusing lidocaine base, the drug was contained in compartments at some distance from the nerve and the cuffs were covered with polystyrene. The completeness of anaesthesia and the functional state of the nerve were tested by stimulating the exposed nerves proximal and distal to the cuff and by observing the behaviour of the muscle prior to killing the animals. The ACh sensitivity was tested by electrophoretic application of ACh from micropipettes and by recording the results changes of the resting membrane potential in individual muscle fibres. The ACh sensitivity was found to be present in the extrajunctional area of all muscle fibres including those displaying miniature end-plate potentials. These was no difference between the behaviour of muscles from rats and rabbits and between the action of lidocaine base and marcaine hydrochloride. Previous reports on the absence of extrajunctional ACh sensitivity in muscles of rabbits whose nerves had been treated by lidocaine base were explained by a relatively rapid loss of the drug from the usual type of nerve cuffs (more than 70% of the drug lost in one day), permitting a premature recovery of the nerves from anaesthesia.     

Excitatory synapses of blue crab gastric mill muscles

Summary Physiological and ultrastructural studies were made of neuromuscular synapses in stomach muscles, especially two gastric mill muscles of the blue crab innervated by neurons of the stomatogastric ganglion. These muscles depolarized and contracted with application of glutamate, but not acetylcholine, whereas the dorsal dilator muscles of the pyloric region depolarized and contracted in acetylcholine, but not in glutamate. Large excitatory postsynaptic potentials (EPSP's) of 5–20 mV were recorded in the gastric mill muscles. At low frequencies of activation, individual synapses released on average about 2 quanta of transmitter for each nerve impulse. Facilitation of EPSP's after a single nerve impulse could be detected for at least 10 s. Synapses were found on enlarged terminals of the motor axon; their contact areas ranged from 0.2 m² up to 3 m². Both electron-lucent, round synaptic vesicles and dense-cored vesicles occurred near these synapses. A possible correlation between contact area of a synapse and output of transmitter, is discussed.Supported by grants from the National Research Council of Canada and the Muscular Dystrophy Association of Canada to H.L. Atwood and C.K. Govind. We thank Kazuko Hay, Eva Yap-Chung and Irene Kwan for technical assistance with electron microscopy and reconstruction of nerve terminals from micrographs     

Dedifferentiation and remodeling of motor endplates following experimental localized lesions of muscle fibers

Local anaesthetics, cardiotoxin and mechanical injuries may cause necrosis of muscle fibres while leaving the motor nerve fibres and their terminals intact. With local injuries to mouse muscles carried out by freezing or cutting we made a point of preserving both the nerve terminals and the muscle fibre portions on which these terminals were located. It was thus possible to follow the changes induced at endplates by these lesions. Within two or three days of the freezing or cutting, the muscle fibres underwent very different degrees of regression of the contractile material and T-system. The neuromuscular junctions also underwent changes, principally affecting their postsynaptic portion, in particular the folds of the subneural apparatus. After dedifferentiation of subsynaptic areas, we observed sprouting of the nerve terminal on muscle fibres which survived the amputation of one end and formed actively new myofibrils. This sprouting restored synaptic connections at the original sites, but with new structural features and correlative changes in the distribution of cholinergic receptors and cholinesterases. It is probable that after a phase of involution followed by a phase of recovery, the injured muscle fibres triggered off the nerve terminal sprouting which led to the remodelling of the endplates.     

Cyclic nucleotides and neuromuscular transmission.

A review of the research on cyclic nucleotides and neuromuscular transmission suggests that cAMP is involved in the release of transmitter from motor nerve endings. Lipid-soluble derivations of cAMP cause depolarization of unstimulated nerve endings and prolong the after potentials of stimulated nerve endings. They also increase the frequency of miniature end plate potentials and increase the quantal content of stimulus evoked end plate potentials. Similar effects are produced by compounds that activate adenylate cyclase or inhibit phosphodiesterase. The responses to the derivatives of cAMP and activators of cyclase are enhanced by inhibitors of phosphodiesterase and prevented by compounds that block the flux of calcium into nerve endings. There is no evidence that suggests that cyclic nucleotides are involved in the postjunctional response to transmitter. Thus, it seems likely that cAMP is involved in the regulation of calcium in motor nerve endings and the exocytosis of transmitter. Additional study should expand our knowledge of neuromuscular transmission and contribute to an understanding of the functions of cyclic nucleotides in other synapses.     

Structure-activity relationship of philanthotoxins--I. Pre- and postsynaptic inhibition of the locust neuromuscular transmission.

1. Like the natural toxin, synthetic delta-philanthotoxin, now called PTX-4.3.3 acts as a reversible postsynaptic open ion-channel blocker of the glutamatergic neuromuscular system of the locust. 2. It also inhibits the high-affinity re-uptake of glutamate in the nerve endings and glial cells. 3. To study the structure-activity relationship, three parts of the PTX-4.3.3 molecule were changed. 4. One of these PTX-analogues, trifluoromethyl-PTX-4.3.3, proved to be a more potent postsynaptic blocker. 5. Moreover, compared with PTX-4.3.3 a delayed recovery period is seen with trifluoromethyl-PTX-4.3.3. 6. A number of PTX-analogues were equipotent to PTX-4.3.3 regarding the inhibition of iontophoretically evoked, postsynaptic glutamate potentials. 7. However, complete inactivation was achieved by reducing the length of the polyamine chain, moreover dideaza-PTX-12 was nearly completely inactive and a reduced activity was seen with dephenol-PTX-4.3.3. 8. A decrease of the decay time constant of glutamate potentials, normally seen by open ion-channel blockers in Con A pretreated preparations, was unaffected during application of the latter two analogues. 9. Possibly these two toxins act as weak receptor antagonists. 10. The presynaptic inhibition of the glutamate re-uptake, seemed to be a very specific property of PTX-4.3.3. Only one of the tested analogues (dehydroxy-PTX-4.3.3) exhibited this capacity.     

Histochemical profiles of guinea-pig intrafusal fibres in normal muscles and after denervation, cordotomy and tenotomy

Synopsis Histochemical profiles of intrafusal fibres were analysed in normal muscles and in denervated, cordotomized and tenotomized preparations. Based on ATPase activity at polar regions, normal intrafusal fibres were classified as (I) ATPase-light fibres showing low or low-moderate activity when pre-incubated in either an acid or alkaline medium; (2) ATPase-dark fibres demonstrating high activity when preincubated in either an acid or alkaline medium and (3) ATPase-reversing fibres displaying low to moderate activity when pre-incubated in an acid medium, but showing high activity when pre-incubated in an alkaline medium. Four weeks after nerve section contrasting responses were seen between intrafusal fibre types. The ATPase-reversing fibres showed large decreases in polar cross-sectional area and NADH-diaphorase (NADH-D) activity, whereas fibres of the ATPase-light and ATPase-dark types were less subject to atrophy and their NADH-D levels were frequently increased. This differential effect suggests that ATPase-reversing fibres are trophically more dependent on neural innervation than ATPase-light and ATPase-dark fibres. After cordotomy and tenotomy no such marked differential responses were noted between fibre types.