BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses

Abstract

Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF-TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory.

Fig. 1.

TrkB receptors are expressed at CA1 synapses in organotypic hippocampal slice cultures. A, The CA1 region was intensely stained with anti-TrkB antibodies.B, The soma and apical dendrites of CA1 pyramidal neurons exhibited TrkB immunoreactivity; punctate staining is shown along the apical dendrites that resembles that of synapses.C, Three representative examples of double immunolabeling with anti-TrkB (green) and anti-synaptobrevin (red) antibodies reveal discrete punctate staining (overlap in yellow) in the apical dendritic region of CA1 that resembles that of synaptic junctions.

Fig. 2.

Representative electron micrographs of synapses on dendritic spines within CA1 stratum radiatum from control (top), BDNF-treated (middle), and K-252a + BDNF-treated (bottom) slice cultures. Reserve pool vesicles, docked vesicles (arrows), and the boundaries of the active zone (arrowheads) are shown.

Fig. 3.

BDNF increases the number of docked vesicles at excitatory CA1 spine synapses, without affecting reserve pool vesicles.A, Histogram plot of the number of docked synaptic vesicles per micrometer of active zone. B, Histogram plot representing the number of reserve pool vesicles per square micrometer of presynaptic terminal. C, Histogram plot of the active zone length. D, Histogram plot of the area of the presynaptic terminal. Data in all panels are means ± SEM (control, n = 98 synapses; BDNF,n = 101 synapses; K-252a + BDNF,n = 53 synapses; an asteriskindicates p < 0.05).

Fig. 4.

The length of the active zone in CA1 asymmetric spine synapses is slightly larger in BDNF-treated slice cultures.A, Representative electron micrograph showing a neuropil field (24 μm²) within CA1 stratum radiatum (from a BDNF-treated slice in this example) that was used for random sampling of synapses. B, Frequency histogram distributions of the active zone length of randomly sampled CA1 spine synapses in 50 nm bins. The distribution of active zone lengths is slightly skewed toward the larger size bins in the BDNF-treated slices. Note also that the BDNF-treated group has a higher number of synapses in the same sampling area (see Results).

Fig. 5.

BDNF increases the frequency of AMPA-mediated mEPSCs without affecting their amplitude. Data are from one control (left), one BDNF-treated (middle), and one K-252a + BDNF-treated (right) neuron.A, Representative AMPA-mediated mEPSCs. BDNF (middle) or K-252a + BDNF (right) did not effect the kinetics or amplitude of AMPA mEPSCs. B, Representative continuous records of membrane currents showing AMPA mEPSC events from control (left), BDNF-treated (middle), and K-252a + BDNF-treated (right) slice cultures. C, Frequency histogram distributions for each of the three depicted cells in 5 pA bins. The total number of events and the frequency of mEPSCs are shown above the frequency histogram distributions.

Fig. 6.

BDNF increases the frequency but not the amplitude of AMPA mEPSCs. A, Cumulative probability distribution of inter-event (mEPSC) interval in all neurons analyzed (6 cells in control, filled circles; 6 cells in BDNF-treated slices,open circles; and 7 cells in K-252a + BDNF-treated slices, filled triangles). B, Cumulative probability distribution of mEPSC amplitude in all neurons analyzed (6 cells in control, filled circles; 6 cells in BDNF-treated slices, open circles; and 7 cells in K-252a + BDNF-treated slices, filled triangles). Note that the difference between the K-252a + BDNF-treated set and the other two groups is not statistically significant at p < 0.05 (see Results). Error bars have been removed for clarity.

Fig. 7.

Most CA1 pyramidal neuron dendritic spines have presynaptic partners. A, Confocal microscopy image of a representative segment of the apical dendrite of a CA1 pyramidal neuron filled with Alexa-594 (red) via the patch electrode during whole-cell recording in a control slice subsequently processed for synaptobrevin immunoreactivity (green). Note that most dendritic spines colocalize with synaptobrevin-positive puncta [yellow (overlap)]. B, Higher magnification view of an Alexa-filled spine with a synaptobrevin-positive presynaptic terminal. C, Scatterplot of the number of spines identified solely by Alexa-filling (left) and the number of synapses identified by Alexa-filling and subsequent immunolabeling of presynaptic terminals with anti-synaptobrevin antibodies (right). The numbers of spines and synapses (normalized per 10 μm of apical dendrite) in each analyzed segment are connected by lines to show the proportion of spines with presynaptic partners (∼65%).

Fig. 8.

BDNF increases spine density in CA1 pyramidal neurons. A, Representative CA1 pyramidal neuron from a BDNF-treated slice filled with Alexa-594 and imaged by confocal microscopy. B, Higher magnification views of representative segments of apical dendrites from control (left), BDNF-treated (middle), and K-252a + BDNF-treated (right) slices used to quantify dendritic spine density. C, Histograms of the number of dendritic spines per 10 μm of CA1 pyramidal neuron apical dendrite. Anasterisk indicates p < 0.05.