The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.
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According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles. (+info)
even-skipped determines the dorsal growth of motor axons in Drosophila.
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Axon pathfinding and target choice are governed by cell type-specific responses to external cues. Here, we show that in the Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and that this expression is necessary and sufficient to direct motor axons into the dorsal muscle field. Previously, it was shown that motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the ventral muscle field. Thus, even-skipped complements the function of islet, and together these two genes constitute a bimodal switch regulating axonal growth and directing motor axons to ventral or to dorsal regions of the muscle field. (+info)
Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle.
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The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers. (+info)
Simultaneous measurement of evoked release and [Ca2+]i in a crayfish release bouton reveals high affinity of release to Ca2+.
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The opener neuromuscular junction of crayfish was used to determine the affinity of the putative Ca2+ receptor(s) responsible for evoked release. Evoked, asynchronous release, and steady-state intracellular Ca2+ concentration, [Ca2+]ss, were measured concomitantly in single release boutons. It was found that, as expected, asynchronous release is highly correlated with [Ca2+]ss. Surprisingly, evoked release was also found to be highly correlated with [Ca2+]ss. The quantal content (m) and the rate of asynchronous release (S) showed sigmoidal dependence on [Ca2+]ss. The slope log m/log [Ca2+]ss varied between 1.6 and 3.3; the higher slope observed at the lower [Ca2+]o. The slope log S/log [Ca2+]ss varied between 3 and 4 and was independent of [Ca2+]o. These results are consistent with the assumption that evoked release is controlled by the sum of [Ca2+]ss and the local elevation of Ca2+ concentration near the release sites resulting from Ca2+ influx through voltage-gated Ca2+ channels (Y). On the basis of the above, we were able to estimate Y. We found Y to be significantly <10 microM even for [Ca2+]o = 13.5 mM. The dissociation constant (Kd) of the Ca2+ receptor(s) associated with evoked release was calculated to be in the range of 4-5 microM. This value of Kd is similar to that found previously for asynchronous release. (+info)
Depression of synaptic efficacy in high- and low-output Drosophila neuromuscular junctions by the molting hormone (20-HE).
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The molt-related steroid hormone, 20-hydroxyecdysone (20-HE), was applied to muscles 6 and 7 of third instar larval of Drosophila melanogaster neuromuscular junction preparations to examine if rapid, nongenomic responses could be observed as was shown recently to occur in crustacean neuromuscular junctions. At a dose of 10 microM, the excitatory junction potentials were reduced in amplitude within minutes. To elucidate the site of action of the hormone, focal-macropatch recordings of synaptic currents were obtained over the neuromuscular junctions. The results showed that the high-output (Is) and the low-output (Ib) motor nerve terminals, which innervate muscles 6 and 7, released fewer synaptic vesicles for each stimulation while exposed to 20-HE. Because the size and shape of synaptic currents from spontaneous releases did not change, the effects of the 20-HE are presynaptic. The rapid effects of this hormone may account in part for the quiescent behavior associated with molts among insects and crustaceans. (+info)
Comparison of local anesthetic activities between optical isomers of cis-1-benzoyloxy-2-dimethylamino-1,2,3,4-tetrahydronaphthalene.
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The optical isomers of cis-1-benzoyloxy-2-dimethylamino-1,2,3,4-tetrahydronaphthalene (YAU-17) were compared for their local anesthetic activity, acute toxicity, spasmolytic activity, and partition coefficient between chloroform and phosphate buffer. 1-YAU-17 was more active than d-YAU-17 in blocking the conduction of action potentials in isolated frog sciatic nerves. The difference in local anesthetic activities between the optical isomers was further substantiated by in vivo tests for corneal anesthesia, intracutaneous anesthesia and sciatic nerve block in quinea-pigs. Similarly, the i.v. injection to mice revealed a higher toxicity for 1-YAU-17 as compared to its d-isomer. In these tests, the potency ratios of the enantiomers ranged from 2 to 4, and the racemate had an intermediate potency. On the contrary, no difference among the compounds was found in their liposolubility, partition coefficient, and spasmolytic activity examined with isolated guinea-pig ileum. These results indicate that the steric factors play an important role in the production of different local anesthetic activities between the optical isomers of YAU-17, and their local anesthetic potency tends to be correlated to their intravenous acute toxicity but not to their spasmolytic activity. (+info)
A glial-neuronal signaling pathway revealed by mutations in a neurexin-related protein.
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In the nervous system, glial cells greatly outnumber neurons but the full extent of their role in determining neural activity remains unknown. Here the axotactin (axo) gene of Drosophila was shown to encode a member of the neurexin protein superfamily secreted by glia and subsequently localized to axonal tracts. Null mutations of axo caused temperature-sensitive paralysis and a corresponding blockade of axonal conduction. Thus, the AXO protein appears to be a component of a glial-neuronal signaling mechanism that helps to determine the membrane electrical properties of target axons. (+info)
Comparison between huperzine A, tacrine, and E2020 on cholinergic transmission at mouse neuromuscular junction in vitro.
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AIM: To compare the effects of huperzine A (Hup A), tacrine, and E2020 on cholinergic transmission at mouse neuromuscular junction in vitro. METHODS: The isolated mouse phrenic nerve-hemidiaphragm preparations were used with the conventional intracellular recording technique. The miniature end-plate potentials (MEPP), the mean quantal content of end-plate potentials (EPP), and the resting membrane potentials of muscle fiber were recorded. RESULTS: Hup A, tacrine, and E2020 at the concentration of 1.0 mumol.L-1 increased the amplitude, time-to-peak, and half-decay time of MEPP in the potencies of E2020 > Hup A > tacrine. Hup A did not significantly change the frequency of MEPP, the appearance of giant MEPP or slow MEPP, the resting membrane potentials, and the mean quantal content of EPP. CONCLUSION: Hup A is a selective and potent cholinesterase inhibitor, by which activity it facilitates the cholinergic transmission at mouse neuromuscular junction, and devoid of pre- and post-synaptic actions. (+info)