BDNF Acutely Modulates Calcium Signalling In Cortical Neurons

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation(LTP). However, different systems show diverse responsiveness to BDNF and some discrepancies have yet to be clarified. In additon, very little is known about the mechanism BDNF modulate synaptic activity and how neuron network activities are modulated by BDNF during development remains to be studied. Further understanding of the molecular and cellular mechanisms underlying the modulation of synaptic activity and synchronized spontaneous neuronal activities by BDNF will not only contribute to the understanding of brain development and brain function but will also lead to the development of potential therapeutic strategies for treatment of abnormal brain activities under pathological conditions. In this study, we applied advanced biophysical techniques, such as patch clamp whole-cell recording, microfluoremetric technique, calcium imaging. Systemic investigations on the effects and the mechanism of BDNF on synaptic transmission and calcium signaling have been carried out in cultured cortical neurons. A novel mechanism of BDNF-induced calcium transients has been proved in cortical neuron; experimental results support the postsynaptic mechanism of BDNF-induced synaptic potentiation; and a novel BDNF-induced long-lasting potentiation of synchronized Ca2+ spikes in cultured cortical networks was found in present study. Main results of the study are as follows: 1. In present study, we found that BDNF rapidly induced potentiation of spontaneous neuron activity of cortical network. Within 3~5 minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as was the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). We also found that a 30 second BDNF application could also rapidly induce an increase in the intracellular Ca2+ concentration in cultured cortical neurons. Above results support the postsynaptic mechanism of BDNF-induced synaptic potentiation. 2. In calcium-free perfusion medium, a substantial calcium signal remained, which disappeared after loading of cortical neurons with 5 μM U-73122, it suggested that the signal partially was caused by calcium mobilization from intracellular stores through the PLC-γ/IP3 pathway. In addition, BDNF-induce Ca2+ transients were partially blocked by Cd2+. In our experiments, we also demonstrated BDNF could rapidly evoke membrane subthreshold depolarization and almost synchronously induce a transient calcium signal in cortical neurons. However, DNF-evoked Ca2+ signals in cortical neuron were altered by neither tetrodotoxin, nor by a cocktail of glutamate receptor blockers CNQX and APV. All data indicate that BDNF-evoked Ca2+ signals result from the influx of extracellular calcium might mainly through voltage-sensitive Ca2+ channels and the calcium mobilization from intracellular stores. 3. Synchronized spontaneous Ca2+ spikes in networked neurons represent periodic burst firing of action potentials, which are believed to play a major role in the development and plasticity of neuronal circuitry. We demonstrate here that cortical neurons in dissociated cultures exhibited synchronized spontaneous Ca2+ spikes in the presence of Mg2+. The synchronized spontaneous Ca2+ spikes are synaptically driven, as it is blocked by tetrodotoxin, and by the glutamate receptor antagonist CNQX/APV. The oscillatory activity is not influenced by GABAA receptor antagonist picrotoxin, suggesting that they entirely rely on glutamatergic neurotransmission. We have also found that these Ca2+ spikes are dependent on an influx of extracellular Ca2+ but are independent of mobilization of Ca2+ from intracellular Ca2+ stores. Ca2+ entry occurred primarily through L-type voltage-gated calcium channels, since nifedipine completely blocked these Ca2+ spikes. In our study, wefound that bath application of BDNF had not any effect on the frequency of the synchronous Ca2+ oscillations in cultured cortical neurons. When we washed out BDNF from bath after the bath application of BDNF (100ng/ml) for 30 minutes, we found whole cortical neuron networks were activated, the frequency of the synchronous Ca2+ oscillations in whole cortical neuron Networks was remarkably potentiated. Furthermore, the BDNF-potentiated Ca2+ oscillations were sustained for 2-3 hours, which depended on the PLC-γ/IP3 pathway and the protein synthesis.