Release of brain-derived neurotrophic factor (BDNF) shapes development and synaptic plasticity of neuronal circuits, thereby controlling brain functions. Analysis of activity-dependent BDNF release can be performed by enzyme-linked immunosorbent assay (ELISA) detection methods or western blot analysis. But these methods require sensitive and specific antibodies directed against the protein of interest and do not allow time-resolved subcellular analysis of protein dynamics. However, knowing the kinetics of activity-dependent secretion as well as subcellular localization of BDNF secretion is important to elucidate the pivotal effects of BDNF on synaptic functions in response to changes in synaptic input activity and activity states of the releasing cells.
Here, we describe an assay which allows for analysis of neuropeptide and protein release on the timescale of several hundred milliseconds and at a resolution of 0.5 μm, thus enabling observation of release from single exocytotic vesicles. To this aim, we constructed plasmids coding for BDNF, tagged at the C-terminus with green fluorescence protein (GFP) or mCherry. Following transfection of dissociated rat hippocampal neurons with one of these plasmids, BDNF secretion can be monitored using wide-field fluorescence live cell imaging. Patterns of electrical activity triggering BDNF secretion were identified by parallel whole-cell patch clamp recordings of releasing neurons. Repetitive depolarizing stimulation as well as the generation of repetitive action potentials (APs) led to a robust BDNF release from neuronal processes. Altogether, these results indicate that BDNF release can be elicited with physiological levels of electrical stimulation.
Activity-dependent release BDNF Fusion protein GFP Live cell imaging Neuron Neurotrophins Secretion Time-lapse fluorescence microscopy
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The authors wish to thank Regina Ziegler and Sabine Eichler for excellent technical assistance and Dr. Amelie Baschwitz for comments on the manuscript.
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