BDNF-induced nitric oxide signals in cultured rat hippocampal neurons: time course, mechanism of generation, and effect on neurotrophin secretion

Abstract

BDNF and nitric oxide signaling both contribute to plasticity at glutamatergic synapses. However, the role of combined signaling of both pathways at the same synapse is largely unknown. Using NO imaging with diaminofluoresceine in cultured hippocampal neurons we analyzed the time course of neurotrophin-induced NO signals. Application of exogenous BDNF, NT-4, and NT-3 (but not NGF) induced NO signals in the soma and in proximal dendrites of hippocampal neurons that were sensitive to NO synthase activity, TrkB signaling, and intracellular calcium elevation. The effect of NO signaling on neurotrophin secretion was analyzed in BDNF-GFP, and NT-3-GFP transfected hippocampal neurons. Exogenous application of the NO donor sodium-nitroprusside markedly inhibited neurotrophin secretion. However, endogenously generated NO in response to depolarization and neurotrophin stimulation, both did not result in a negative feedback on neurotrophin secretion. These results suggest that a negative feedback of NO signaling on synaptic secretion of neurotrophins operates only at high intracellular levels of nitric oxide that are under physiological conditions not reached by depolarization or BDNF signaling.

Keywords: BDNF; PSD95; TrkB; neurotrophins; nitric oxide; peptide secretion; synaptic plasticity.

Figure 1

Time course of BDNF-induced NO signals in hippocampal neurons. Microcultures of rat hippocampal neurons (15–18 DIV) were loaded with the fluorescent NO indicator DAF, and changes in fluorescence intensity of DAF were monitored using time-lapse confocal microscopy. (A) Images of BDNF (100 ng/ml, bath application starting at 0 s)-induced NO signal in a single hippocampal neuron at time points as indicated. Note the increase of NO in the soma and proximal dendrites. (B) Average (n = 9 cells from 3 experiments) NO increase induced by bath applied BDNF (100 ng/ml) vs. negative control (continuously superfused with HBS). Vertical arrow indicates time point of drug application, ^*p < 0.01. (C) Averaged parallel NO increase in soma vs. dendrites in the same individual cells (n = 5; different cells than shown in B). (D) Average (n = 5 cells) NO increase induced by SNP (100 μM), used as positive control, ^**p < 0.001 vs. negative control. (E) Mean BDNF-induced DAF fluorescence intensity 10 min after start of stimulation. Drug application (100 ng/ml BDNF, 300 μM L-NMMA, 10 μM L-NAME) as indicated. Note the complete inhibition of BDNF-induced NO signals in the presence of NOS inhibitors. ^***p < 0.001 vs. control; ### p < 0.001 BDNF + L–NAME vs. BDNF. Errors bars represent s.e.m.

Figure 2

Activation of Trk receptors and intracellular Ca²⁺ elevation are required for BDNF-induced NO generation. Cultured hippocampal neurons (15–18 DIV) were loaded with the NO indicator DAF, and DAF fluorescence of wells was monitored with a plate reader. (A) Time course of average fluorescence intensities (n = 4) is shown for drug treatments (at 1 min, 1 μM Ionomycin, 300 μM L-NMMA) as indicated. (B) Average DAF fluorescence 20 min after treatments as indicated. Ionomycin served as positive control. ^*p < 0.01 compared with negative control, ^**p < 0.001 compared with BDNF positive control. Note the almost complete block of BDNF-induced NO signals by BAPTA (10 μM) and k252a (200 nM). Error bars represent s.e.m. (C) NO signals induced by BDNF, NGF, NT-3, NT-4, respectively (100 ng/ml, each; average DAF fluorescence 20 min after treatment as indicated). Note the independence of BDNF-induced NO signaling from inhibition of glutamatergic transmission (D/APV = 10 μM DNQX, 50 μM APV). (D) Inhibition of L–VGCCs with nifedipine (10 μM, average DAF fluorescence after 20 min treatment) did not affect BDNF-induced NO signals. ^*p < 0.05, ^**p < 0.01 compared with negative control. Error bars represents s.e.m.

Figure 3

Measurement of synaptic release of neurotrophins. (A) Hippocampal neurons were co-transfected at 8-DIV with BDNF-GFP and PSD95-DsRed. Co-localization of both proteins was monitored at 10–11 DIV. Boxed areas on the left are shown at higher magnification on the right. Postsynaptic vesicle clusters of BDNF-GFP (green) were identified by co-localization with PSD95-DsRed (Red). (B) Time course of postsynaptic BDNF-GFP and NT-3-GFP release in response to depolarization with elevated K⁺ (50 mM, 300s). (C) Residual fluorescence of postsynaptic BDNF-GFP and NT-3-GFP after 300 s depolarization. Experiments were performed in the presence of 10 μM DNQX, 200 μM D,L-APV, and 10 μM gabazine, to avoid secondary effects via transmitter secretion. ^**p vs control < 0.0001. Error bars represent s.e.m.

Figure 4

Somatic vs. dendritic NO signals induced by 50 mM K⁺-induced stimulation. Hippocampal neurons (10–12 DIV) were loaded with the fluorescent NO indicator DAF-FM, and changes in DAF fluorescence intensity were monitored using time-lapse video microscopy. (A) Typical example of DAF-FM loaded hippocampal neurons following stimulation with 50 mM K⁺ used for analysis of fluorescence intensity in the soma as shown in (B). (B) time course of DAF fluorescence upon stimulation with 50 mM K⁺ starting at 200 s. Average DAF fluorescence intensity in hippocampal neurons exposed to 50 mM K⁺ solution under control conditions (n = 25 cells; open circles) and after pre-incubation (10 min) in the presence of 300 μM L-NMMA (n = 32; black squares). (C,D) Typical DAF-FM loaded hippocampal neuron with color coded somatic and dendritic regions analyzed in (D). Note the simultaneous increase in DAF fluorescence in somatic (red), proximal (green), and distal dendrites (blue). (E) Subcellular resolution of DAF-FM signals following local pressure-application of high K⁺ (50 mM). High K⁺ solution was applied via a small pipette (left hand image) directed toward the dendrite of a hippocampal neuron. The suction pipette was positioned such that direct depolarization of the soma was avoided. (F) Normalized DAF fluorescence of color coded regions marked in E indicates simultaneous NO-generation in dendritic compartments, whereas no increase was detected in the cell soma. (G) Average peak increase in DAF fluorescence in 5 independent experiments as shown in (C,D). (H) Average peak increase in DAF fluorescence in 5 independent experiments as shown in (E,F). Data represent means ± s.e.m.

Figure 5

NO dependent modulation of synaptic release of BDNF and NT-3. Hippocampal neurons were transfected with BDNF-GFP (BDNF) or NT-3-GFP (NT-3) and monitored for neurotrophin secretion. (A,C) Averaged depolarization-induced (50 mM K⁺) release of NTs vs. negative control. (B,D) Mean residual fluorescence 300s after stimulation. (A,B) Preincubation with the NO donor SNP (100 μM, 5 min) reduced depolarization-induced secretion of neurotrophins. (C,D) Preincubation and subsequent superfusion with the NOS inhibitor L–NMMA (300 μM, 5 min) during depolarization did not change the amount of neurotrophins that was released. Experiments were performed in the presence of 10 μM DNQX, 200 μM D,L-APV, and 10 μM gabazine, to avoid secondary effects via transmitter secretion. Negative control cells were superfused with HBS throughout. ^*p vs. control < 0.05, ^**p vs control < 0.005. Error bars represent s.e.m.