Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice

Abstract

Brain-derived neurotrophic factor (BDNF) promotes long-term potentiation (LTP) at hippocampal CA1 synapses by a presynaptic enhancement of synaptic transmission during high-frequency stimulation (HFS). Here we have investigated the mechanisms of BDNF action using two lines of BDNF knockout mice. Among other presynaptic impairments, the mutant mice exhibited more pronounced synaptic fatigue at CA1 synapses during high-frequency stimulation, compared with wild-type animals. Quantitative analysis of CA1 synapses revealed a significant reduction in the number of vesicles docked at presynaptic active zones in the mutant mice. Synaptosomes prepared from the mutant hippocampus exhibited a marked decrease in the levels of synaptophysin as well as synaptobrevin [vesicle-associated membrane protein (VAMP-2)], a protein known to be involved in vesicle docking and fusion. Treatment of the mutant slices with BDNF reversed the electrophysiological and biochemical deficits in the hippocampal synapses. Taken together, these results suggest a novel role for BDNF in the mobilization and/or docking of synaptic vesicles to presynaptic active zones.

Fig. 1.

Pronounced synaptic fatigue in CA1 synapses during high-frequency stimulation in BDNF knockout mice. a, Examples of EPSPs elicited by a train of HFS (100 Hz) in hippocampal slices from CD1/BDNF mice. Note the significant synaptic fatigue in +/− and −/− mice. b, Summary of synaptic fatigue in CD1/BDNF mice. The slope of the 40th EPSP in the train is presented as the percentage of the first EPSP slope.n = number of recordings. c, Summary of synaptic fatigue in BL/6/BDNF mice.

Fig. 2.

Overall changes in synaptic responses during HFS in CA1 synapses of CD1/BDNF mice. a, EPSP slopes were plotted against the number of the stimulus in the 100 Hz train for each of the synapses, and the integrated areas under the plots were calculated for each synapse and compared among three genotypes. *, Significantly different; ANOVA test with post hoc PLSD Fischer’s test. n = 8 for each genotype.b, Rate of synaptic fatigue in +/+, +/−, and −/− synapses. Normalized EPSP slopes were plotted against the number of the stimulus during HFS. The plot for each genotype was fitted with a single exponential curve, and rate constants were obtained.n = 8 for each genotype.

Fig. 3.

Impairment of post-tetanic potentiation (PTP) in CD1/BDNF mice. a, Examples of EPSPs before and 1 min after tetanus in different genotypes (separated by double slash). b, Summary of PTP in +/+, +/−, and −/− mice. EPSP slopes monitored within a 3 min time window after tetanus (2 × 1 sec, 100 Hz) were averaged, and PTP is expressed as a percentage of the baseline EPSP slope (collected during the 20 min before tetanus). *, Significantly different from +/+; ANOVA, p < 0.005. n = 5, 5, and 4 for +/+, +/−, and −/−, respectively.

Fig. 4.

Impairment of paired-pulse facilitation (PPF) at short interpulse intervals in CD1/BDNF mice. a, Sample EPSP traces during PPF at 10 and 50 msec interpulse intervals. b, Plot of PPF at different interpulse intervals. *, Significantly different from +/+; ANOVA,p < 0.005. n = 5, 5, and 4 for +/+, +/−, and −/−, respectively.

Fig. 5.

Electron micrographs of excitatory synapses on CA1 dendritic spines in hippocampal stratum radiatum from +/+, +/−, and −/− CD1/BDNF mice. Examples of presynaptic terminals and adjacent dendritic spines displaying normal gross structural features in the three genotypes. Arrows point to a representative example of a morphologically docked vesicle as is defined in the text; arrowheads mark the edges of the active zone/postsynaptic density complexes. Note that +/− and −/− mice have fewer docked vesicles at the active zones.

Fig. 6.

Quantitative analysis of synaptic vesicles in excitatory synapses in BDNF mutant mice. a, Number of docked vesicles per micrometer of active zone length in the three genotypes. Data from CD1/BDNF and those from BL/6/BDNF mutant mice were combined. b, Number of reserve pool vesicles per presynaptic terminal in the three genotypes. n = number of synapses; N = number of animals.

Fig. 7.

Analysis of synaptic proteins in homogenates of whole hippocampal preparations and in hippocampal synaptosomes from BDNF knockout mice. a, Synaptic proteins detected in whole hippocampal homogenates from BL/6/BDNF mice. The hippocampi were dissected from a pair of +/+ and +/− mice, homogenized, and subject to Western blot analysis using antibodies against specific synaptic proteins. b, Synaptic proteins detected in whole hippocampal preparations (right, homogenate) and synaptosomes (left) from CD1/BDNF mice. Similar Western blot analysis was performed. Note the significant reduction in the levels of synaptobrevin (VAMP-2) and synaptophysin only in synaptosomal preparations, but not of other synaptic proteins. Because synaptotagmin, SNAP25, and syntaxin all show the same levels in the same gel, there is no need to include loading controls in this figure.c, Relative levels of the synaptic proteins in the synaptosomes, quantified by scanning the blots and comparing the bands with standard curves. In each experiment, hippocampal synaptosomes were prepared from a large litter of CD1/BDNF mice (2–3 months old), with three to four +/+ and three to four +/− of the same gender, and Western blots using the same synaptosomal preparation were repeated several times. Four such experiments were performed, and each experiment was repeated two to three times. Data are pooled and presented as a percentage of the wild-type levels.

Fig. 8.

Effect of acute exposure to BDNF on synaptic fatigue and the levels of synaptobrevin and synaptophysin in BDNF mutant slices. a, Synaptic fatigue was induced by a train of 1 sec, 100 Hz stimulation in hippocampal slices from either +/− or −/− CD1/BDNF mice. The slices were then treated with recombinant BDNF (2 nm) for 3 hr. HFS responses in +/− and −/− slices before and after BDNF treatment were normalized to that in +/+ slices (n = 21). N = 3, 2, and 2 for +/+, +/−, and −/−, respectively. b, Examples of Western blots showing the levels of synaptobrevin and synaptophysin in synaptosomes from +/+ and +/− slices and +/− slices plus BDNF. Slices from +/− CD1/BDNF mice were treated with BDNF (2 nm) for 3 hr. Synaptosomes were prepared and synaptic proteins were detected the same way as described in Figure 7. Note that the same levels of actin were detected in all three lanes, suggesting that equal amounts of proteins are loaded. c, Quantification of the relative levels of synaptobrevin and synaptophysin in synaptosomes prepared from +/− slices treated with and without BDNF. Three experiments using independent synaptosomal preparations were performed. Each experiment was repeated two to three times, and data were pooled and presented as a percentage of the wild-type levels.