Fast excitatory synaptic transmission mediated by nicotinic acetylcholine receptors in Drosophila neurons. (1/107)

Difficulty in recording from single neurons in vivo has precluded functional analyses of transmission at central synapses in Drosophila, where the neurotransmitters and receptors mediating fast synaptic transmission have yet to be identified. Here we demonstrate that spontaneously active synaptic connections form between cultured neurons prepared from wild-type embryos and provide the first direct evidence that both acetylcholine and GABA mediate fast interneuronal synaptic transmission in Drosophila. The predominant type of fast excitatory transmission between cultured neurons is mediated by nicotinic acetylcholine receptors (nAChRs). Detailed analysis of cholinergic transmission reveals that spontaneous EPSCs (sEPSCs) are composed of both evoked and action potential-independent [miniature EPSC (mEPSC)] components. The mEPSCs are characterized by a broad, positively skewed amplitude histogram in which the variance is likely to reflect differences in the currents induced by single quanta. Biophysical characteristics of the cholinergic mEPSCs include a rapid rise time (0.6 msec) and decay (tau = 2 msec). Regulation of mEPSC frequency by external calcium and cobalt suggests that calcium influx through voltage-gated channels influences the probability of ACh release. In addition, brief depolarization of the cultures with KCl can induce a calcium-dependent increase in sEPSC frequency that persists for up to 3 hr after termination of the stimulus, illustrating one form of plasticity at these cholinergic synapses. These data demonstrate that cultured embryonic neurons, amenable to both genetic and biochemical manipulations, present a unique opportunity to define genes/signal transduction cascades involved in functional regulation of fast excitatory transmission at interneuronal cholinergic synapses in Drosophila.  (+info)

Rapid and reversible effects of activity on acetylcholine receptor density at the neuromuscular junction in vivo. (2/107)

Quantitative fluorescence imaging was used to study the regulation of acetylcholine receptor (AChR) number and density at neuromuscular junctions in living adult mice. At fully functional synapses, AChRs have a half-life of about 14 days. However, 2 hours after neurotransmission was blocked, the half-life of the AChRs was now less than a day; the rate was 25 times faster than before. Most of the lost receptors were not quickly replaced. Direct muscle stimulation or restoration of synaptic transmission inhibited this process. AChRs that were removed from nonfunctional synapses resided for hours in the perijunctional membrane before being locally internalized. Dispersed AChRs could also reaggregate at the junction once neurotransmission was restored. The rapid and reversible alterations in AChR density at the neuromuscular junction in vivo parallel changes thought to occur in the central nervous system at synapses undergoing potentiation and depression.  (+info)

Protein kinase C activity regulates slow myosin heavy chain 2 gene expression in slow lineage skeletal muscle fibers. (3/107)

Expression of the slow myosin heavy chain (MyHC) 2 gene defines slow versus fast avian skeletal muscle fiber types. Fetal, or secondary, skeletal muscle fibers express slow MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow MyHC 2 gene expression in vitro is regulated by a combination of innervation and cell lineage. This pattern of gene expression was indistinguishable from the pattern observed in vivo in that it was restricted to innervated muscle fibers of slow muscle origin. We show here that slow MyHC 2 gene expression in the slow muscle fiber lineage is regulated by protein kinase C (PKC) activity. Inhibition of PKC activity induced slow MyHC 2 gene expression, and the capacity to express the slow MyHC 2 gene was restricted to muscle fibers of slow muscle (medial adductor) origin. Fast muscle fibers derived from the pectoralis major did not express significant levels of slow MyHC 2 with or without inhibitors of PKC activity. This differential expression pattern coincided with different inherent PKC activities in fast versus slow muscle fiber types. Furthermore, over-expression of an unregulated PKCalpha mutant suppressed slow MyHC 2 gene expression in muscle fibers of the slow lineage. Lastly, denervation of skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in vivo. These results provide a mechanistic link between innervation, an intracellular signaling pathway mediated by PKC, and expression of a muscle fiber type-specific contractile protein gene. Dev Dyn 1999;216:177-189.  (+info)

Formation of cholinergic synapses between dissociated sympathetic neurons and skeletal myotubes of the rat in cell culture. (4/107)

Sympathetic principal neurons, dissociated from superior cervical ganglia of newborn rats, were plated into cultures containing rat skeletal myotubes formed from previously plated primary myoblasts. Electrophysiological evidence is presented that the neurons developed cholinergic synapses with the myotubes. In addition, the neurons developed cholinergic synapses with each other as previously reported [O'Lague et al. (1974) Proc. Nat. Acad. Sci. USA 71, 3602-3606]. The acetylcholine receptors of myotubes differed from those of the neurons in their sensitivities to curare and hexamethonium, in a manner expected of adult muscle and ganglionic receptors. alpha-Bungarotoxin blocked synaptic transmission from neuron to myotube, but not from neuron to neuron in the same culture.  (+info)

Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: effects of cAMP-pathway mutations. (5/107)

We describe a novel bioassay system that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila "giant" neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and alpha-bungarotoxin, but not by TTX and Cd(2+), suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis.  (+info)

Dual effects of ether on end-plate currents. (6/107)

1. The effects of diethyl ether (ether) on miniature end-plate currents (m.e.p.c.s) and on acetylcholine-activated end-plate channels were measured in toad sartorius muscle fibres with voltage-clamp and extracellular recording techniques. 2. At low concentrations (less than 20 mM) either made m.e.p.c.s decay faster than normal. At high concentrations (greaster than 40 mM), the decay of m.e.p.c.s was slower than normal. With all concentrations, the cecay remained exponential with single time constant, tau D. 3. At low concentrations ether did not affect the growth phase of m.e.p.c.s and only slightly reduced the amplitude of m.e.p.c.s. At the higher concentrations, the growth phase was slowed and m.e.p.c.s were significantly reduced in amplitude. 4. Ether at all concentrations (5--70 mM) reduced end-plate channel lifetime, the effect increasing with ether concentration. Ether did not significantly alter the elementary channel conductance or the actylcholine null (reversal) potential. 5. Curare reduced tau D which had been prolonged by high concentrations of ether. Ether itself at high concentrations caused a reduction in tau D increased by neostigmine. It is proposed that high concentrations of either inhibit acetylcholine hydrolysis by acetylcholinesterase. 6. The effect of ether in reducing end-plate channel lifetime and reducing m.e.p.c. amplitude, without significantly altering the normal voltage and temperature sensitivity of channel lifetime, is consistent with the proposal that either reduces the stability of open end-plate channels.  (+info)

The effects of prolonged repetitive stimulation in hemicholinium on the frog neuromuscular junction. (7/107)

1. Cutaneous pectoris nerve-muscle preparations from the frog were stimulated for prolonged periods in solutions with curare alone, curare and hemicholinium no. 3 (HC-3), or curare and glucose plus choline. End-plate potentials (e.p.p.s) and miniature end-plate potentials (m.e.p.p.s) were recorded intracellularly. Black widow spider venom (BWSV) was applied to determine the degree of depletion of the transmitter stores. 2. The ultrastructure of the neuromuscular junctions was studied in the electron microscope. Some of the preparations were fixed immediately at the end of the period of stimulation and others were fixed about an hour after BWSV had been applied. In some experiments horseradish peroxidase (HRP) was present during the period of stimulation and the fixed tissue was treated to reveal the distribution of the tracer. 3. The amplitude of the e.p.p. fell rapidly to almost zero during 2 hr of stimulation at 2/sec in 100 muM HC-3 and little recovery occurred during a subsequent hour of rest. About 2-7 times 10-5 quanta were secreted. The e.p.p.s usually persisted throughout the period of stimulation in the other solutions and 2-2-6 times as much transmitter was secreted. 4. When BWSV was applied immediately at the end of the period of stimulation in HC-3, almost no m.e.p.p.s were discharged and only small m.e.p.p.s were discharged when the venom was applied after an hour of rest. 5. When BWSV was applied to unstimulated terminals that had been bathed in HC-3, or to terminals that had been stimulated and rested for an hour in glucose plus choline, m.e.p.p.s of nearly normal amplitude were discharged. 6. Terminals stimulated for 2 hr at 2/sec in 100 muM HC-3 contained a normal complement of synaptic vesicles and a large proportion of vesicles were labelled with HRP when the tracer was present during the period of stimulation. 7. BWSV induced the almost complete depletion of vesicles from terminals that had been stimulated in HC-3. 8. Depletion of vesicles also occurred when terminals were stimulated for 20 min at 10/sec after they had been previously stimulated for 2 hr at 2/sec in HC-3. These terminals showed extensive infolding of the axolemma and they contained swollen mitochondria. 9. These results indicate that stimulation in HC-3 depletes terminals of their store of transmitter but not of their population of vesicles and that vesicles empty of transmitter can fuse with and reform from the axolemma of the nerve terminal.  (+info)

The action of acetylcholine antagonists on amino acid responses in the frog spinal cord in vitro. (8/107)

1 The isolated hemisected frog spinal cord has been used to study the action of acetylcholine antagonists on amino acid responses by means of sucrose gap recording. 2 Primary afferents and motoneurones were shown to contain few, if any, cholinoceptors, since acetylcholine and carbachol responses were essentially abolished when synaptic transmission was blocked with magnesium ions or when action potentials were blocked by tetrodotoxin. 3 Curare antagonized the gamma-aminobutyric acid (GABA) and beta-alanine depolarizations of primary afferents and hyperpolarizing action of these amino acids on motoneurones. Nicotine also antagonized beta-alanine depolarizations and to a small extent GABA depolarizations of primary afferents. These actions are similar to but weaker than those obtained previously with picrotoxin. 4 Atropine selectively antagonized beta-alanine depolarizations of primary afferents and blocked beta-alanine and glycine hyperpolarizations of motoneurones. GABA responses were entirely resistant to the action of atropine. These actions are similar to but 50 times weaker than those obtained previously with strychnine. 5 Dihydro-beta-erythroidine, tetraethylammonium, and gallamine were entirely ineffective in antagonizing amino acid responses. Since these agents are known to block the dorsal root potential elicited by ventral root stimulation but have no effect on the amino acid responses of primary afferents, it is evident that a cholinergic step is involved in this pathway.  (+info)

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