BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses

Abstract

Exerting its actions pre-, post- and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines - a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity.

Figure 1. BDNF persistently accelerates the initial phase of evoked release from presynaptic terminals on dendritic spines of eGFP-transfected cultured hippocampal neurons

A, initial raw fluorescent intensities of hippocampal presynaptic terminals from control and brain-derived neurotrophic factor (BDNF)-treated cultures (3 h and 3 days) following FM1-43 staining with a strong stimulus (900 APs at 10 Hz) loading ∼80% of the total recycling pool, as estimated by high K⁺ stimulation (Student's t test, P > 0.05). B, initial raw fluorescent intensities of hippocampal excitatory presynaptic terminals on dendritic spines of eGFP-transfected control and BDNF-treated neurons (3 days) following FM4-64 staining with a weaker stimulus (300 APs at 10 Hz) loading ∼40% of the total recycling pool, as estimated by high K⁺ stimulation (Student's t test, P > 0.05). C, either 3 h or 3 days exposure to BDNF accelerated FM1-43 destaining evoked by field electrical stimulation (1200 APs at 10 Hz). D, time-lapse confocal images of individual hippocampal excitatory presynaptic nerve terminals (FM4-64, red puncta) overlapping onto dendritic spines of a control postsynaptic neuron (eGFP, green). Time-lapse images were acquired for baseline, as well as during the delivery of the field stimulation protocol used to evoke synaptic vesicle exocytosis. Note the decrease in brightness of red puncta across time (left to right) corresponding to a decrease in FM4-64 fluorescence as a result of exocytosis. E, background-subtracted line plots of mean normalized pixel intensities during baseline acquisition, and during field stimulation, for terminals from control (red) and BDNF-treated cultures (green). The area in the box is enlarged in the plot to the right to highlight the effect of BDNF on FM destaining during the initial phase of the stimulation protocol. Asterisks indicate significant differences (ANOVA followed by Scheffé's procedure for multiple comparisons, P < 0.05).

Figure 2. BDNF persistently enhances the evoked and spontaneous destaining rate of FM1-43 from a rapidly recycling pool (RRP) in CA1 stratum radiatum of acute hippocampal slices visualized by multiphoton microscopy

A, schematic representation of the stimulation of CA3 afferent fibres and recording of extracellular fEPSPs in CA1 stratum radiatum superimposed on a bright-field image of an acute hippocampal slice. The yellow box represents the area imaged by multiphoton microscopy. B, multiphoton excitation (840 nm) fluorescence images of FM1-43 loaded into RRP vesicles by hyperosmotic shock (25 s, 800 mosmol l⁻¹ aCSF with sucrose) within stratum radiatum of area CA1 (yellow box in A) of a control hippocampal slice. Note the row of CA1 pyramidal cell bodies at the top of the image. The areas within the solid white boxes are shown at higher magnification in the panels below. Note the tip of the stimulating electrode in the middle left of the top panel (as a triangular non-fluorescent object). The activity-dependent FM1-43 destaining of terminals within the area denoted by the dotted white line is shown in D. C, initial raw fluorescent intensities of RRP-labelled terminals from control and BDNF-treated slices following FM1-43 staining using sucrose to load the RRP or high K⁺ to load the total recycling pool of vesicles (Student's t test, *P < 0.05). D, activity-dependent destaining of FM1-43 from RRP-labelled terminals within the dotted box shown in B (numbers represent time in minutes; 0′ is the beginning of the unloading stimulation, i.e. 10 Hz 2 s bursts every 30 s). E, time course of stimulus-evoked FM1-43 destaining from RRP-labelled terminals in control (open circles) versus BDNF-treated (filled circles) slices showing enhanced vesicular release from the RRP after BDNF (background-corrected and normalized averaged pixel intensity within multiple ROIs centred over stable fluorescent puncta). The vertical bars indicate the stimulation to the afferent fibres consisting of 10 Hz 2 s bursts every 30 s throughout and synchronized with image acquisition. The enhanced rate of activity-dependent release from the RRP after BDNF exposure is completely prevented by coapplication of the Trk inhibitor k-252a (filled triangles). F, time course of spontaneous FM1-43 destaining from RRP-labelled terminals in control (open circles) versus BDNF-treated (filled circles) slices showing enhanced vesicular release from the RRP after BDNF. The enhancement of spontaneous FM1-43 destaining is also blocked by coapplication of k-252a (filled triangles).

Figure 3. BDNF increases the probability of transmitter release at CA3–CA1 synapses

A, superimposed evoked EPSCs (no failures) recorded in CA1 pyramidal neurons from a control slice at three different concentrations of extracellular Ca²⁺ (4, 2 and 1 mm). Panels below the traces show the frequency distribution of EPSC amplitudes at each extracellular Ca²⁺ concentration fitted with Gaussian distributions. B, variance-mean analysis assuming a binomial model of synaptic transmission (Silver, 2003). Plot of the normalized variance of EPSC amplitudes versus the estimated release probability fitted by single parabolas in control (open circles) and BDNF-treated (filled circles) slices. EPSCs were evoked at different extracellular Ca²⁺ concentrations in the presence of a submaximal concentration of the AMPA receptor antagonist DNQX (100 nm) to prevent receptor saturation. As indicated by the overlapping parabolic fits from BDNF-treated and control cells, there were no significant differences in either quantal amplitude or number of independent release sites (Student's t test P > 0.05). C, mean probability of release (P_r) values derived from the parabolic fits plotted as a function of [Ca²⁺]_o. P_r was enhanced by BDNF pre-exposure (filled circles) at all three extracellular Ca²⁺ concentrations (ANOVA for multiple measures, *P < 0.05; see also Table 1).

Figure 4. The BDNF scavenger TrkB-IgG prevents the enhancement of FM1-43 release from the RRP after LTP induction

A, average initial raw FM1-43 fluorescence intensities of RRP-labelled terminals from control slices, and in slices after LTP induction in the absence and presence of the BDNF scavenger TrkB-IgG. FM1-43 staining was performed using sucrose to selectively load the RRP (Student's t test, *P < 0.05, **P < 0.001). B, time course of evoked FM1-43 destaining from the RRP in control slices (open circles) versus slices where LTP was induced (filled circles), showing the enhanced vesicular release from the RRP after LTP induction in area CA1. The vertical bars indicate the stimulation to the afferent fibres consisting of 10 Hz 2 s bursts every 30 s throughout and synchronized with image acquisition. The enhanced rate of activity-dependent release from the RRP after LTP induction is completely prevented by application of the BDNF scavenger TrkB-IgG before the LTP-inducing stimuli (filled triangles).