RESUMO
The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both - 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.
RESUMO
The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25-30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25-30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30-40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.