Activity-dependent release of endogenous BDNF from mossy fibers evokes a TRPC3 current and Ca2+ elevations in CA3 pyramidal neurons

Abstract

Multiple studies have demonstrated that brain-derived neurotrophic factor (BDNF) is a potent modulator of neuronal structure and function in the hippocampus. However, the majority of studies to date have relied on the application of recombinant BDNF. We herein report that endogenous BDNF, released via theta burst stimulation of mossy fibers (MF), elicits a slowly developing cationic current and intracellular Ca(2+) elevations in CA3 pyramidal neurons with the same pharmacological profile of the transient receptor potential canonical 3 (TRPC3)-mediated I(BDNF) activated in CA1 neurons by brief localized applications of recombinant BDNF. Indeed, sensitivity to both the extracellular BDNF scavenger tropomyosin-related kinase B (TrkB)-IgG and small hairpin interference RNA-mediated TRPC3 channel knockdown confirms the identity of this conductance as such, henceforth-denoted MF-I(BDNF). Consistent with such activity-dependent release of BDNF, these MF-I(BDNF) responses were insensitive to manipulations of extracellular Zn(2+) concentration. Brief theta burst stimulation of MFs induced a long-lasting depression in the amplitude of excitatory postsynaptic currents (EPSCs) mediated by both AMPA and N-methyl-d-aspartate (NMDA) receptors without changes in the NMDA receptor/AMPA receptor ratio, suggesting a reduction in neurotransmitter release. This depression of NMDAR-mediated EPSCs required activity-dependent release of endogenous BDNF from MFs and activation of Trk receptors, as it was sensitive to the extracellular BDNF scavenger TrkB-IgG and the tyrosine kinase inhibitor k-252b. These results uncovered the most immediate response to endogenously released--native--BDNF in hippocampal neurons and lend further credence to the relevance of BDNF signaling for synaptic function in the hippocampus.

Fig. 1.

Release of endogenous brain-derived neurotrophic factor (BDNF) from mossy fibers (MFs) activates MF-I_BDNF, a nonselective cationic current in CA3 pyramidal neurons. A: representative examples of AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) evoked in CA3 pyramidal neurons by MF stimulation, before and after application of DCG-IV. B: representative examples of slow currents evoked by theta burst stimulation (TBS) of MFs in CA3 pyramidal neurons in the presence of antagonists of iono- and metabotropic glutamate receptors as well as GABA_A receptors (left). The MF-evoked current (MF-I_BDNF) in the same cell was significantly reduced by the extracellular BDNF scavenger tropomyosin-related kinase B (TrkB)-IgG, but not by human control IgG (right). C: input-output curve of slow currents evoked by TBS of MFs.

Fig. 2.

Vesicular Zn²⁺ released during MF stimulation does not contribute to MF-I_BDNF. A: Ca-EDTA did not affect MF-I_BDNF. B: neither Ca-EDTA nor ZnCl₂ affected I_BDNF, the membrane currents evoked by direct application of recombinant BDNF to apical dendrites (in TTX). C: CA3 pyramidal neurons transfected with TRPC3 small hairpin interference RNA (shRNA) show smaller currents than untransfected CA3 neurons in the same slice and cells transfected with a random shRNA (which show currents similar to untransfected cells).

Fig. 3.

MF-I_BDNF is associated with elevations in dendritic Ca²⁺ levels. A, left: representative example of Ca²⁺ signals evoked during MF stimulation in the presence of TTX, iono- and metabotropic glutamate, and GABA receptor antagonists. Ca²⁺ elevations occurred throughout the apical dendrites and the soma. (right), The BDNF scavenger TrkB-IgG (1 μg/ml) prevented Ca²⁺ elevations during MF stimulation in the antagonist cocktail. B: quantification of the changes in peak fura-2 ratios (360 nm/380 nm) during MF stimulation.

Fig. 4.

A single brief TBS of MFs causes a BDNF-dependent depression of excitatory postsynaptic currents in CA3 pyramidal neurons. A: representative example illustrating the enduring depression of AMPAR-mediated EPSCs after TBS of MFs. Inset: representative EPSCs before (1) and 50 min after (2) TBS. B: representative example illustrating the enduring depression of N-methyl-d-aspartate receptor (NMDAR)-mediated EPSCs after TBS of MFs. Inset: representative EPSCs before (1) and 40 min after (2) TBS. C: TBS of MFs did not affect the NMDAR/AMPAR ratio. D: plots of the fractional changes in coefficient of variation (CV², r) vs. the synaptic gain factor (f) after TBS of MFs. Note that all points fall to the right of the diagonal (r = f).

Fig. 5.

The enduring depression of NMDAR-mediated EPSCs was sensitive to the BDNF scavenger TrkB-IgG and the receptor tyrosine kinase inhibitor k-252b. A: representative example illustrating the prevention of MF-TBS-induced depression of NMDAR-mediated EPSCs by bath application of TrkB-IgG. B: representative example illustrating the prevention of MF-TBS-induced depression of NMDAR-mediated EPSCs by intracellular application of k-252b.