Backpropagating Action Potentials Trigger Dendritic Release of BDNF during Spontaneous Network Activity

Abstract

Brain-derived neurotrophic factor (BDNF) is a major regulator of activity-dependent synapse development and plasticity. Because BDNF is a secreted protein, it has been proposed that BDNF is released from target neurons in an activity-dependent manner. However, direct evidence for postsynaptic release of BDNF triggered by ongoing network activity is still lacking. Here we transfected cultures of dissociated hippocampal neurons with green fluorescent protein (GFP)-tagged BDNF and combined whole-cell recording, time-lapse fluorescent imaging, and immunostaining to monitor activity-dependent dendritic release of BDNF. We found that spontaneous backpropagating action potentials, but not synaptic activity alone, led to a Ca 2ϩ -dependent dendritic release of BDNF-GFP. Moreover, we provide evidence that endogenous BDNF released from a single neuron can phosphorylate CREB (cAMP response element-binding protein) in neighboring neurons, an important step of immediate early gene activation. Therefore, together, our results support the hypothesis that BDNF might act as a target-derived messenger of activity-dependent synaptic plasticity and development.

GABABReceptor Activation Triggers BDNF Release and Promotes the Maturation of GABAergic Synapses

“…After transfection, the cultures were incubated at 30°C in 5% CO 2 . Immunostaining confirmed that BDNF-GFP is targeted to the dendrite and packed into secretory granules of the regulated pathway of secretion (Kuczewski et al, 2008b).…”

“…Neurons from postnatal day 0 rat hippocampus were dissociated using trypsin and plated on coverslips coated with polyethylenimine as previously described (Kuczewski et al, 2008b). Eleven days after plating, neurons were transfected with cDNAs coding for BDNF-GFP (gift from Dr. V. Lessmann, Institute of Physiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany) according to the OZ Biosciences protocol (Buerli et al, 2007).…”

“…Real-time imaging was performed as previously described (Kuczewski et al, 2008b). Fluorescence intensity was measured from dendritic regions containing clusters of BDNF-GFP with ImageJ software after subtraction of background fluorescence.…”

“…The procedure for surface BDNF-GFP immunostaining was similar to that previously described (Kuczewski et al, 2008b). Briefly, after being washed in ACSF, the living cultures were incubated at 4°C for 1 h in the presence of an anti-GFP antibody (10 g/ml; Molecular Probes).…”

“…The procedure for phospho-CREB activation and immunocytochemistry was similar to that previously described (Kuczewski et al, 2008b). Briefly, 1 d before stimulation (at 13 DIV) one-half of the culture medium was changed to MEM with 2% B27 supplement.…”