Single-cell characterization of retrograde signaling by brain-derived neurotrophic factor

Abstract

Brain-derived neurotrophic factor (BDNF) is a key regulator of hippocampal synaptic plasticity in the developing and adult nervous system. It can be released from pyramidal neuron dendrites in an activity-dependent manner and has therefore been suggested to serve as a signal that provides the retrograde intercellular communication necessary for Hebbian plasticity and hippocampal-dependent learning. Although much has been learned about BDNF function by field stimulation of hippocampal neurons, it is not known whether moderate action potential-independent depolarization of single cells is capable of releasing sufficient BDNF to influence transmission at individual synapses. In this study, we show directly at the single-cell level that such modulation can occur. By using K-252a, anti-BDNF antibody, and interruption of regulated release, we confirm a model in which postsynaptic depolarization elicits calcium-dependent release of BDNF that diffuses retrogradely and enhances presynaptic transmitter release.

Figure 1.

Postsynaptic depolarization elicits an increase in presynaptic release probability. A, Hippocampal neurons in tissue culture. Whole-cell patch-clamp recordings were made from pyramidal-like neurons. Presynaptic activity was assessed by monitoring mEPSC frequency. The patch electrode indicates an example of the type of cell selected for recording. B, Representative traces of mEPSCs taken at different times during a recording at holding potentials of −40 mV (traces on left) or −70 mV (traces on right). Depolarization of the cell being recorded from influences the rate of mEPSCs, an assay of presynaptic transmitter release probability. Calibration: 50 pA, 200 ms. C, Average time course of mEPSC frequency with a holding potential of −40 mV (n = 8; •) or −70 mV (n = 7; ○). Event frequency gradually increased in depolarized neurons but remained stable in neurons that were held at the relatively negative potential. The holding potential of −40 mV caused a significant elevation of event frequency compared with the holding potential of −70 mV (p < 0.05, two-tailed Student's t test). Error bars indicate ± SEM. Hold pot, Holding potential.

Figure 2.

The effect of postsynaptic depolarization is mimicked by BDNF, is prevented by selective antagonists, and does not occur when elevation of intracellular calcium or regulated release is blocked. A, Bath perfusion of 20 ng/ml BDNF reproduced the effect of postsynaptic depolarization in cells that were recorded at a holding potential of −70 mV (n = 9; •). B, K252a prevented the effect of depolarization if it was included in the bath solution (n = 4; ▵) but not if it was only included in the pipette solution, thus affecting only the cell being recorded from (n = 6; ▴). The latter finding implicates binding of BDNF to presynaptic trkB receptors in the production of the depolarization effect. C, Bath perfusion of an anti-BDNF function-blocking antibody (n = 5; ▾) or increasing the concentration of EGTA in the pipette solution to 10 mm (n = 5; gray squares) prevented the effect of postsynaptic depolarization. D, Neurons transfected with Box1 continue to show a postsynaptic depolarization-induced increase in mEPSC frequency (n = 5; ♦), whereas neurons transfected with Box2+3 do not (n = 5; ◇). Error bars indicate ± SEM.

Figure 3.

Summary of the different manipulations on the presynaptic action of released BDNF. A, Plot of average event frequency at 22–27 min during the recordings normalized to baseline. K252a (Bath), anti-BDNF, and Box2+3 were significantly different from Box1 (p < 0.01, two-tailed Student's t test), whereas BDNF and K252a (Int) were not (p > 0.3 and p > 0.6, respectively, two-tailed Student's t test). B, Diagrammatic summary of the proposed mechanism by which postsynaptic depolarization leads to an increase in presynaptic transmitter release. Postsynaptic depolarization evokes calcium-dependent release of BDNF from dendrites (1). BDNF diffuses retrogradely from the postsynaptic to the presynaptic terminals (2) and activates presynaptic trkB receptors (3). This in turn elicits an increase in vesicle release probability (4). These steps were confirmed by interrupting regulated release, with a BDNF scavenging antibody to block diffusion and by blocking presynaptic trkB function. Error bars indicate ± SEM. NT vesicle, Neurotransmitter-containing vesicle.