Intracellular Ca2+ stores and Ca2+ influx are both required for BDNF to rapidly increase quantal vesicular transmitter release

Abstract

Brain-derived neurotrophic factor (BDNF) is well known as a survival factor during brain development as well as a regulator of adult synaptic plasticity. One potential mechanism to initiate BDNF actions is through its modulation of quantal presynaptic transmitter release. In response to local BDNF application to CA1 pyramidal neurons, the frequency of miniature excitatory postsynaptic currents (mEPSC) increased significantly within 30 seconds; mEPSC amplitude and kinetics were unchanged. This effect was mediated via TrkB receptor activation and required both full intracellular Ca(2+) stores as well as extracellular Ca(2+). Consistent with a role of Ca(2+)-permeable plasma membrane channels of the TRPC family, the inhibitor SKF96365 prevented the BDNF-induced increase in mEPSC frequency. Furthermore, labeling presynaptic terminals with amphipathic styryl dyes and then monitoring their post-BDNF destaining in slice cultures by multiphoton excitation microscopy revealed that the increase in frequency of mEPSCs reflects vesicular fusion events. Indeed, BDNF application to CA3-CA1 synapses in TTX rapidly enhanced FM1-43 or FM2-10 destaining with a time course that paralleled the phase of increased mEPSC frequency. We conclude that BDNF increases mEPSC frequency by boosting vesicular fusion through a presynaptic, Ca(2+)-dependent mechanism involving TrkB receptors, Ca(2+) stores, and TRPC channels.

Figure 1

BDNF rapidly increases mEPSC frequency. (a) Representative trace demonstrating the effect of BDNF on mEPSC frequency recorded in CA1 pyramidal cells. BDNF was applied at a concentration of 100 ng/mL from a Picrospritzer-controlled pipette aimed at CA1 striatum radiatum in a 14 div slice culture. Insets show representative mEPSCs at higher time resolution. (b) Left: representative running average plot of mEPSC frequency as a function of time (sec); right: mean mEPSC frequency before and after BDNF application. (c) Top: tepresentative probability distributions of mEPSC amplitudes prior to and following application of BDNF; bottom: cumulative probability distributions for amplitude and interevent intervals prior to and following application of BDNF.

Figure 2

The increase in mEPSC frequency requires TrkB receptor activation. (a) Top: representative trace showing the BDNF effect in a CA1 pyramidal cell in the presence of the receptor tyrosine kinase inhibitor k-252a (200 nM); left: representative running average plot of mEPSC frequency in the presence of k252a; right: mean frequency of mEPSCs before and after BDNF in the continuous presence of k252a. (b) Top: representative trace recorded showing the BDNF effect in a CA1 pyramidal cell dialyzed with k252b (200 nM), a membrane impermeable inhibitor of the Trk receptor family; left: running average plot of mEPSC frequency in a representative k252b-loaded neuron; right: mean frequency of mEPSCs before and after BDNF in k252b-loaded neurons.

Figure 3

BDNF requires Ca²⁺ influx and intracellular Ca²⁺ mobilization to increase mEPSC frequency. (a) Top: representative trace from a CA1 pyramidal cell in the absence of extracellular Ca²⁺ prior to and following BDNF application; left: representative running average plot of mEPSC frequency in the absence of extracellular Ca²⁺; right: mean frequency of mEPSCs before and after BDNF in the absence of extracellular Ca²⁺. (b) Top: representative trace from a CA1 pyramidal cell in the presence of the SERCA pump inhibitor thapsigargin (1 μM) prior to and following BDNF application; left: representative running average plot of mEPSC frequency in the presence of thapsigargin; right: mean frequency of mEPSCs before and after BDNF in the continuous presence of thapsigargin. (c) Top: representative trace from a CA1 pyramidal cell loaded with BAPTA prior to and following BDNF application; left: running average plot of mEPSC frequency in a representative BAPTA-loaded neuron, right: mean frequency of mEPSCs before and after BDNF in BAPTA-loaded neurons.

Figure 4

Inhibiting TRPC channels prevented the BDNF-induced increase in mEPSC frequency. Top: representative trace from a CA1 pyramidal cell in the presence of the SOC/TRPC channel inhibitor SKF96365 (30 μM) prior to and following BDNF application; left: representative running average plot of mEPSC frequency in the presence of SKF96365; right: mean frequency of mEPSCs before and after BDNF in the continuous presence of SKF96365.

Figure 5

BDNF caused destaining of FM dyes in the presence of TTX. (a) Left: representative image of FM1-43 labeled puncta in hippocampal slice cultures by multiphoton excitation microscopy (840 nm excitation); right: BDNF caused destaining of FM1-43 labeled puncta with a τ = 506 ± 62 sec (8.4 min); n = 6 slices. (b) Left: representative image of FM2-10 labeled puncta in hippocampal slice cultures by multiphoton excitation microscopy (840 nm excitation), right: BDNF caused destaining of FM2-10 labeled puncta with a τ = 403 ± 118 sec (6.7 min); n = 4 slices.