ABSTRACT
INTRODUCTION: Brain-derived neurotrophic factor (BDNF) inhibits neuromuscular junction (NMJ) maturation. In this study we investigated the underlying molecular mechanisms of this process. METHODS: We used a patch-clamp technique to measure spontaneous synaptic currents (SSCs) from innervated muscle cells in Xenopus nerve-muscle cocultures. RESULTS: In the presence of Ca(2+)/calmodulin-dependent kinase (CaMK) inhibitor KN93, SSC amplitude (226.3 ± 26.5 pA), frequency (30.9 ± 10.1 events/min), and percentage of bell-shaped amplitude distributions (47.1%) were reversed to control levels (286.7 ± 48.2 pA, 26.2 ± 5.8 events/min, and 47.1%, respectively). Depletion of intracellular Ca(2+) by BAPTA-AM or thapsigargin had similar reversal effects to KN93. In addition, cotreatment with both 2-APB (IP3 receptor inhibitor) and TMB-8 (ryanodine receptor inhibitor) also reversed the inhibitory effects of BDNF, as shown by the physiological parameters. CONCLUSIONS: CaMK mediates the inhibitory effects of BDNF on NMJ maturation. Ca(2+) released from intracellular stores through either IP3 receptors or ryanodine receptors regulates neurotrophic actions on NMJ maturation.
Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Calcium/metabolism , Intracellular Fluid/metabolism , Neuromuscular Junction/embryology , Neuromuscular Junction/metabolism , Animals , Intracellular Fluid/drug effects , Neuromuscular Junction/drug effects , Organ Culture Techniques , XenopusABSTRACT
The development of neuromuscular junction (NMJ) is initiated by motor axon's contact with the skeletal muscle cell that is followed by synaptic maturation. Previous studies showed that brain-derived neurotrophic factor (BDNF) enhanced motoneurons' survival and growth but significantly inhibited synaptogenesis. Here, we report that chronic application of BDNF resulted in inhibition in the maturation process both physiologically and morphologically. The response to BDNF was mediated by its cognate receptor TrkB as the effects were abolished by Trk receptor inhibitor K252a. Protein kinase A (PKA) inhibitor reversed the effects of BDNF in inhibiting NMJ maturation. These results indicate that BDNF suppresses NMJ maturation through cAMP-PKA signaling pathway. Together with the previous studies, these results suggest that BDNF suppresses NMJ formation and maturation despite its effects in enhancing neuronal survival and growth.
Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Neuromuscular Junction/drug effects , Animals , Brain-Derived Neurotrophic Factor/metabolism , Carbazoles/pharmacology , Cells, Cultured , Indole Alkaloids/pharmacology , Neuromuscular Junction/embryology , Neuromuscular Junction/physiology , Receptor, trkB/antagonists & inhibitors , XenopusABSTRACT
The neuromuscular junction (NMJ) is established through initial contact of motor neuron axon with a skeletal muscle cell and the subsequent synaptic maturation. Previous studies have shown that cyclic AMP (cAMP) enhanced spinal neurons' survival and growth but inhibited synaptogenesis. Here, we find that elevating intracellular cAMP level of presynaptic neurons prevented NMJs from maturation both physiologically and morphologically. Importantly, cytosolic Ca(2+) is essential for the inhibitory effects of cAMP on NMJ maturation. We show that depletion of intracellular Ca(2+) store, rather than extracellular Ca(2+), abolished the cAMP-dependent inhibition of synaptic maturation. Taken together, we demonstrate that Ca(2+) released from intracellular Ca(2+) stores regulates neurotrophic actions on NMJ maturation.
