Postsynaptic secretion of BDNF and NT-3 from hippocampal neurons depends on calcium calmodulin kinase II signaling and proceeds via delayed fusion pore opening

Abstract

The mammalian neurotrophins (NTs) NGF, BDNF, NT-3, and NT-4 constitute a family of secreted neuronal growth factors. In addition, NTs are implicated in several forms of activity-dependent synaptic plasticity. Although synaptic secretion of NTs has been described, the intracellular signaling cascades that regulate synaptic secretion of NTs are far from being understood. Analysis of NT secretion at the subcellular level is thus required to resolve the role of presynaptic and postsynaptic NT secretion for synaptic plasticity. Here, we transfected cultures of dissociated rat hippocampal neurons with green fluorescent protein-tagged versions of BDNF and NT-3, respectively, and identified NT vesicles at glutamatergic synapses by colocalization with the cotransfected postsynaptic marker PSD-95 (postsynaptic density-95)-DsRed. Depolarization-induced secretion of BDNF and NT-3 was monitored with live cell imaging. Direct postsynaptic depolarization with elevated K+ in the presence of blockers of synaptic transmission allowed us to investigate the signaling cascades that are involved in the postsynaptic NT vesicle secretion process. We show that depolarization-induced postsynaptic NT secretion is elicited by Ca2+ influx, either via L-type voltage-gated calcium channels or via NMDA receptors. Subsequent release of Ca2+ from internal stores via ryanodine receptors is required for the secretion process. Postsynaptic NT secretion is inhibited in the presence of KN-62 ([4(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester) and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide), indicating a critical dependence on the activation of alpha-calcium-calmodulin-dependent protein kinase II (CaMKII). The cAMP/protein kinase A (PKA) signaling inhibitor Rp-cAMP-S impaired NT secretion, whereas elevation of intracellular cAMP levels was without effect. Using the Trk inhibitor k252a, we show that NT-induced NT secretion does not contribute to the NT release process at synapses, and BDNF does not induce its own secretion at postsynaptic sites. Release experiments in the presence of the fluorescence quencher bromphenol blue provide evidence for asynchronous and prolonged fusion pore opening of NT vesicles during secretion. Because fusion pore opening is fast compared with compound release, the speed of NT release seems to be limited by diffusion of NTs out of the vesicle. Together, our results reveal a strong dependence of activity-dependent postsynaptic NT secretion on Ca2+ influx, Ca2+ release from internal stores, activation of CaMKII, and intact PKA signaling, whereas Trk signaling and activation of Na+ channels is not required.

Figure 1.

Investigation of postsynaptic secretion of BDNF and NT-3. A, Hippocampal neurons were cotransfected at 8 DIV with BDNF-GFP and PSD-95-DsRed. Colocalization of both proteins were monitored at 10–11 DIV. B, Boxed area in A shown at higher magnification. Postsynaptic vesicle clusters of BDNF (green) were identified by colocalization with PSD-95 (red). C, Time course of BDNF release in response to depolarization with elevated K⁺ (50 mm; 300 s). Pictures show postsynaptic BDNF-GFP vesicle clusters at indicated time points (0 s, start of depolarization; compare D). Note the gradual decrease of intracellular BDNF-GFP after the start of depolarization (+150 s; +300 s). D, Time course of intracellular fluorescence of regions of interest in C. Note the decrease in intracellular fluorescence during stimulation (bar). E, Postsynaptic BDNF secretion in response to different depolarization paradigms (red, 6 × 10 s elevated K⁺ at 10 s intervals; green, 300 s K⁺). Comparable degrees of NT secretion after 120 s are evident.

Figure 2.

Bromphenol blue quenching of GFP fluorescence reveals fast initial fusion pore opening of BDNF-GFP vesicles. Hippocampal neurons were transfected with GFP-tagged BDNF, and depolarization-induced (50 mm K⁺, 300 s; postsynaptic receptors blocked with 10 μm DNQX, 50 μm d,l-APV, and 50 μm gabazine) decrease in intracellular GFP fluorescence was monitored. A, Average time course of fluorescence decay in the absence/presence of the green fluorescence quencher BPB (2 mm in all superfusion solutions). In the absence of BPB, the fluorescence decay reflects release of BDNF-GFP, but in the presence of BPB, the decay represents the kinetics of first fusion pore openings of the BDNF-GFP vesicles (compare Results). Note the fast onset of first fusion pore openings compared with the delayed and less pronounced release of BDNF-GFP. B, Two representative series of pictures (dashed blue box in A) for a cell measured in the presence of BPB. Note the sudden and complete loss of fluorescence of a single vesicle (blue arrows) within a 10 s imaging interval. C, Two representative series of pictures (dashed red box in A) for another cell measured in the absence of BPB. Note the graded fluorescence decay of single vesicles (red arrows) even during a 200 s imaging interval. The sudden BPB-dependent quenching of intravesicular BDNF-GFP fluorescence explains the faster and more complete decrease of the average data for BPB cells in A. Scale bars, 2 μm.

Figure 3.

Prolonged or repetitive fusion pore opening of BDNF-GFP vesicles. All experimental details are as in Figure 2. A, Fluorescence intensity of single BDNF vesicles in the color-coded circles in B and C. Horizontal bars indicate superfusion conditions. B, C, Two image sequences (time windows indicated by stippled brackets) of the vesicles analyzed in A. The repeated disappearance/reappearance of the vesicles marked in red and green, after wash-in/washout of BPB, indicates prolonged and/or repetitive fusion pore opening during the depolarization. The transient increase in fluorescence intensity of the “green” vesicle at +100 s is caused by reduced quenching of GFP fluorescence after neutralization of intragranular pH through the fusion pore (Brigadski et al., 2005). The unchanged fluorescence of the “yellow” vesicle reflects absence of fusion pore opening for this vesicle. D, Recording as in A of single vesicle and vesicle cluster fluorescence in response to elevated K⁺ solution in the presence of BPB. Note the large scatter in delays until first fusion pore opening for individual vesicles from the same dendrite. Scale bar, 2 μm.

Figure 4.

Depolarization-induced postsynaptic NT secretion depends on Ca²⁺ influx. Hippocampal neurons were transfected with GFP-tagged BDNF and NT-3, and depolarization-induced (50 mm K⁺, 300 s; postsynaptic receptors blocked with 10 μm DNQX, 50 μm d,l-APV, and 50 μm gabazine) postsynaptic secretion of NTs was measured as the decrease in intracellular fluorescence intensity. A, Change in BDNF-GFP secretion after consecutive depolarizations of the same cell in the absence/presence of extracellular Ca²⁺ (2 mm). B, Time course of secretion of BDNF-GFP and NT-3-GFP. Preincubation with the L-type Ca²⁺ channel antagonist nifedipine (50 μm; 10 min) significantly inhibited (p < 0.0001, at t = 300 s) secretion of both NTs. C, Residual intracellular fluorescence after 300 s stimulation for conditions as indicated. **p < 0.0001 compared with negative control (neg. con). Note the strong dependence of BDNF and NT-3 secretion on the activity of L-type VGCCs. Error bars represent SEM.

Figure 5.

Activation of NMDA receptors can trigger postsynaptic secretion of BDNF-GFP. Experimental details are as in Figure 4. A, Time course of secretion of BDNF-GFP after application of 300 μm NMDA in saline containing 2 mm Ca²⁺ and 0 mm Mg²⁺. These experiments were performed in the presence of DNQX (10 μm), gabazine (50 μm), and nifedipine (10 μm) to allow for selective observation of NMDAR-mediated release. B, Time course of elevated K⁺ induced secretion of BDNF in the absence or presence of tetrodotoxin (TTX; 0.8 μm). Error bars represent SEM. neg. control, Negative control.

Figure 6.

Dependence of NT secretion on intracellular Ca²⁺ stores and CaMKII. Hippocampal neurons were transfected with BDNF-GFP (BDNF) and monitored for postsynaptic neurotrophin secretion as in Figure 5. A, C, Time course of depolarization-induced (50 mm K⁺) release of BDNF-GFP. B, D, Average residual BDNF-GFP fluorescence after 300 s depolarization. B, Preincubation with thapsigargine (10 μm; 10 min) or CPA (3 μm, 30 min) to deplete internal Ca²⁺ stores or with ryanodine (80 μm; 30 min) to block Ca²⁺-induced Ca²⁺ release from internal stores significantly inhibited NT secretion (*p < 0.0001). D, Preincubation with 10 μm KN-62 or 10 μm KN-93 to inhibit CaMKII significantly inhibited NT secretion. For KN-62, data from BDNF and NT-3 release were pooled (*p < 0.01). neg. control, Negative control; rel., relative.

Figure 7.

Unaltered presynaptic transmitter secretion during pharmacological treatments. A, Presynaptic terminals of hippocampal neurons were loaded with FM 4-64 at 10 DIV. Presynaptic secretion of FM 4-64 was elicited with elevated K⁺ (50 mm; 40 s). B, Boxed area in A shown at higher magnification. Pictures show FM 4-64 fluorescence in presynaptic terminals at indicated time points (0 s, start of depolarization; compare C). C, Average time course of FM release in untreated (control) and treated neurons (50 μm nifedipine). Note the similar time courses of FM secretion, indicating unaltered presynaptic secretion after the drug treatment also used in NT secretion experiments. D, Average residual FM 4-64 fluorescence after 40 s depolarization for different treatments as indicated. All fluorescence values are given relative to the fluorescence levels at the start of the depolarization. Note that the different drug treatments did not significantly change presynaptic release of transmitters, indicating intact synaptic transmitter release under our recording conditions. neg. control, Negative control; rel., relative.

Figure 8.

Postsynaptic cAMP/PKA activity gates secretion of neurotrophins. Hippocampal neurons were transfected with BDNF-GFP (BDNF) and monitored for neurotrophin secretion as described previously. A, C, Averaged depolarization-induced (50 mm K⁺) release of BDNF versus negative control. B, D, Mean residual fluorescence 300 s after stimulation. A, Preincubation with the PKA activator 8-Br-cAMP (100 μm; 5 min) did not change depolarization-induced BDNF secretion. B, Application of 8-Br-cAMP (100 μm) neither induced BDNF secretion (left) nor changed the amount of released BDNF in response to depolarization (right) (*p < 0.005). C, D, Preincubation with the PKA inhibitor Rp-cAMP-S (100 μm; 5 min) significantly inhibited and delayed BDNF secretion. Experiments were performed in the presence of 10 μm DNQX, 50 μm d,l-APV, and 50 μm gabazine. Note that inhibition of basal levels of PKA signaling delays secretion of BDNF-GFP. neg. control, Negative control; rel., relative.

Figure 9.

Absence of NT-induced NT secretion at postsynaptic sites of hippocampal synapses. Hippocampal neurons were transfected with BDNF-GFP (BDNF) and monitored for neurotrophin secretion as described above. A, Averaged depolarization-induced (50 mm K⁺) or BDNF-induced (100 ng/ml) release of BDNF-GFP versus negative control. All recordings were performed in the presence of 10 μm DNQX, 50 μm d,l-APV, and 50 μm gabazine to block postsynaptic ionotropic receptors. When BDNF was applied, the extracellular solution contained nifedipine (10 μm) to abolish indirect effects of BDNF on L-type Ca²⁺ channels and to monitor selectively release that was elicited by BDNF-induced release of Ca²⁺ from internal stores. B, C, Average depolarization-induced (50 mm K⁺) residual fluorescence, 300 s after start of depolarization, of GFP-tagged BDNF or NT-3 versus control. Preincubation with the Trk kinase inhibitor k252a (200 nm; 30 min) had no significant effect (p > 0.05) on depolarization-induced NT secretion.

Figure 10.

Scheme of the signaling cascades involved in postsynaptic secretion of NTs. Influx of extracellular Ca²⁺, either via L-type VGCCs or via NMDA receptors, is required for depolarization induced secretion. Amplification of the initial Ca²⁺ influx through Ca²⁺-induced Ca²⁺ secretion via ryanodine receptors is necessary to observe NT secretion. CaMKII signaling is pivotal for the induction of NT secretion. Basal levels of PKA activation “gate” NT secretion. Activation of voltage-gated Na⁺ currents is not necessary to observe NT release (data not shown). NTs do not sustain their own release from postsynaptic sites (data not shown).