Botulinum neurotoxin type A (BoNT/A) paralyses muscles by blocking acetylcholine (ACh) release from motor nerve terminals. Although highly toxic, it is used clinically to weaken muscles whose contraction is undesirable, as in dystonias. The effects of an injection of BoNT/A wear off after 3-4 months so repeated injections are often used. Recovery of neuromuscular transmission is accompanied by the formation of motor axon sprouts, some of which form new synaptic contacts. However, the functional importance of these new contacts is unknown. Using intracellular and focal extracellular recording we show that in the mouse epitrochleoanconeus (ETA), quantal release from the region of the original neuromuscular junction (NMJ) can be detected as soon as from new synaptic contacts, and generally accounts for > 80% of total release. During recovery the synaptic delay and the rise and decay times of endplate potentials (EPPs) become prolonged approximately 3-fold, but return to normal after 2-3 months. When studied after 3-4 months, the response to repetitive stimulation at frequencies up to 100 Hz is normal. When two or three injections of BoNT/A are given at intervals of 3-4 months, quantal release returns to normal values more slowly than after a single injection (11 and 15 weeks to reach 50% of control values versus 6 weeks after a single injection). In addition, branching of the intramuscular muscular motor axons, the distribution of the NMJs and the structure of many individual NMJs remain abnormal. These findings highlight the plasticity of the mammalian NMJ but also suggest important limits to it.

Background: Septins serve as scaffolds for membrane-associated protein complexes. Results: Knockdown of septin-2 or disruption of septin assembly/disassembly impairs interactions between exocytic proteins and inhibits late steps of exocytosis. Conclusion: Septins undergo dynamic reorganization to facilitate localized and timely interactions between exocytosis-essential proteins. Significance: Both the presence of septin-2 and active reorganization of septin oligomers are required for exocytosis.

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Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.

The effects of calcium and strontium on the quantal content of nerve-evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2-0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca 2+ ] o was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca 2+ ] o . Hence, extracellular calcium affected mainly the late, 'asynchronous', portion of phasic release. The average quantal content grew exponentially from 0.09 ± 0.01 to 1.04 ± 0.07 with the increase in [Ca 2+ ] o without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2-1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) (25 lmol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N¢,N¢-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 lmol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms. Keywords: BAPTA-AM, calcium, EGTA-AM, quantal release, strontium, time course of the evoked quanta release.

Uniquantal endplate currents (EPCs) were recorded simultaneously at the proximal, central and distal parts of the frog neuromuscular synapse, and their minimal synaptic latencies, latency dispersions and sensitivity to noradrenaline, cAMP and protein kinase A inhibition were measured. The latency dispersion was highest in the proximal part (P 90 = 1.25 ms); it decreased to P 90 = 0.95 ms in the central part and to P 90 = 0.75 ms (60 % of the proximal part) in the distal part. In the proximal parts of the long neuromuscular synapse, stimulation-evoked EPCs with long release latencies were eliminated when the intracellular cAMP was increased by b1 activation by noradrenaline, by the permeable analogue db-cAMP, by activation of adenylyl cyclase or by inhibition of cAMP hydrolysis. This makes the evoked release more compact, and the amplitude of the reconstructed multiquantal currents increases. Protein kinase A is a target of this regulation, since a specific inhibitor, Rp-cAMP, prevents the action of cAMP in the proximal parts and increases the occurrence of long-latency events in the distal parts of the synapse. Our results show that protein kinase A is involved in the timing of quantal release and can be regulated by presynaptic adrenergic receptors. Journal of Physiology (2002), 538.3, pp. 837-848 DOI: 10.1013/jphysiol.2001.012752 © The Physiological Society 2002 www.jphysiol.org 1984 Shakiryanova et al. 1994), non-uniform spontaneous and evoked release (Zefirov, 1983; Mallart, 1984; D'Alonzo & Grinnell, 1989) as well as different structural characteristics of the proximal and distal endplate regions (Davey & Bennett, 1982; Robitaille & Tremblay, 1987). We therefore estimated the latencies of EPCs simultaneously at the proximal, central and distal parts of the synapse and found substantial differences between these parts in minimal synaptic latencies, latency dispersions and sensitivity to NA, cAMP manipulations and PKA inhibition. Preliminary results with cAMP in the proximal part of the synapse have already been presented as a short report (Bukharaeva et al. 2000). METHODS Animals and drugsThe experimental preparation, bathing solutions and focal extracellular recording technique have been described in a previous paper (Bukharaeva et al. 1999). Briefly, experiments were carried out on the cutaneous pectoris muscle from the frog Rana ridibunda from November to April. Animals were anaesthetized with ether before being stunned and double-pithed. The preparations were pinned to a translucent chamber and superfused with (m): NaCl 113.0, KCl 2.5, CaCl 2 0.2, NaHCO 3 3.0, and MgCl 2 4.0. The pH was 7.3 at 20.0 ± 0.3°C. The Animal Care and Use Committee of the Institute of Physiology, Czech Academy of Sciences, approved the protocol.The following drugs were used (Sigma, St Louis, MO, USA): NA, dibutyryl cAMP, adenosine 3,5-monophosphothioate (Rp-cAMP), forskolin, and 3-isobutyl-1-methylxanthine (IBMX). The drugs were added to the superfusing solution, and the measurements were started 20 min after drug applic...

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca(2+), and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129-132, 2005; Bukharaeva et al., J Neurochem 100:939-949, 2007).

Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end-plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC(50) = 1.1 microm) or by carbachol acting on muscarinic M2 receptors (EC(50) = 1.8 microm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross-talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor-specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1-[[2-[(diethylamino)methyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one) (AFDX-116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8-(p-sulfophenyl)theophylline (8-SPT) or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane-delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca(2+) influx. We propose that the negative cross-talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine.