Figure 1.

Spontaneous network activity triggers BDNF-GFP release. a–f, Overlapped images showing intracellular GFP fluorescence (green) and secreted BDNF-GFP detected using anti-GFP antibody (red) under nonpermeable conditions. a, Hippocampal neurons transfected with GFP alone. b–f, Neurons transfected with BDNF-GFP. b, Control condition. c, In the presence of TTX (1 μm). d, Stimulated with 4-AP (50 μm). e, Stimulated with 4-AP (50 μm) in the presence of NBQX (5 μm), d-APV (40 μm), and bicuculline (10 μm), indicated in the figure as “antago.” f, Stimulated with 4-AP (50 μm) in the presence of NBQX, d-APV, bicuculline, and TTX (1 μm). Top, Merged picture of both fluorescence channels of neurons transfected with the BDNF-GFP construct (green) and immunocytochemistry staining using an antibody against the GFP (red). Bottom, Quantification of the surface-bound BDNF-GFP on the transfected neuron (yellow signal); the plots are a three-dimensional representation of the mean gray level values. g, Quantitative analysis of surface-bound GFP comparing BDNF-GFP-transfected neurons in different ACSF conditions (n = 4 different cultures, 4 neurons per culture in each condition). h, Average firing rate of the neurons in the different conditions, measured in cell-attached configuration (n = 5 neurons in each condition). Right, Representative electrophysiological traces for each condition. The green signal produced by released BDNF-GFP is not visible in the images presented here because of the low laser intensity used to avoid saturation of the green signal in the transfected neurons. The red signal in the untransfected cell is attributable to the BDNF-GFP secreted by the transfected neurons that bound to membrane TrkB receptors of the neighboring cells. Antago in e, f, and h stands for NBQX (5 μm), APV (40 μm), and bicuculline (10 μm). Ctr, Control. In this and following figures, * indicates p < 0.05 compared with control condition.