BDNF-GFP CONTAINING SECRETORY GRANULES ARE LOCALIZED IN THE VICINITY OF SYNAPTIC JUNCTIONS OF CULTURED CORTICAL-NEURONS

The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin- 3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS , PNS), In addition, exogenous application of neurotrophins has recent ly been found to modulate synaptic transmission in the rodent CNS, How ever, to provide evidence for a role of neurotophins as endogenous fas t acting modulators of synaptic transmission, the synaptic localizatio n and secretion of neurotrophins needs to be shown. We have now constr ucted a fusion protein consisting of N-terminal BDNF (the most abundan t neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localizatio n of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS -7 cells revealed that the cellular localization in the trans-Golgi ne twork (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from properties of untagged BDNF , Upon transient transfection of primary I-at cortical neurons, BDNF-G FP was found in secretory granules of the regulated pathway of secreti on, as indicated by colocalization with the secretory granule marker s ecretogranin II. BDNF-GFP vesicles were found in the neurites of trans fected neurons with a pattern reminiscent of the localization of endog enous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were f ound predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immu nocytochemical staining of synaptic terminals with synapsin I antibodi es revealed that the density of BDNF-GFP vesicles is elevated in the v icinity of synaptic junctions, indicating that BDNF is localized appro priately to function as an acute modulator of synaptic transmission. T hese data suggest that BDNF-GFP will be a useful tool to investigate s ynaptic release of BDNF during physiological synaptic stimulation, and will thereby allow us to elucidate the participation of neurotrophin release in activity dependent synaptic plasticity.