Journal: The Journal of Neuroscience

Article Title: Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

doi: 10.1523/JNEUROSCI.1776-05.2005

Figure Lengend Snippet: Intact processing and biological function of GFP-tagged neurotrophins. GFP-tagged NTs were expressed in COS7 cells, and cell lysates and supernatants were analyzed 2 d after transfection. A, Anti-GFP Western blot reveals dominant presence of the NT precursors in the cell lysates (top, arrows, NT-4 precursor with lower MW than the other NTs) and of the correctly processed mature NTs in the COS cell supernatants (bottom, arrow). The horizontal marks at the left indicate the position of GFP. B, NT-induced fiber outgrowth of PC12 cells via overexpressed TrkB and endogenously expressed TrkA receptors, respectively. PC12 cells were cotransfected at 1 DIV with TrkB and GFP plasmids and were incubated for 3 d with supernatants from COS cells overexpressing (for 2 d) GFP-tagged BDNF, NGF, NT-3, NT-4, or wt GFP, respectively, or were incubated with rhBDNF. C, Quantification of NT-induced fiber outgrowth of PC12 cells after 3 d of incubation with COS cell supernatants and recombinant (rec.) neurotrophins. **Significantly different from all NTs with p < 0.01. D, Anti-BDNF Western blot of COS cells expressing wt BDNF and BDNF-GFP, respectively, and of human recombinant BDNF. The blot indicates comparable amounts of wt BDNF and BDNF-GFP in the supernatants used in E. E, PC12 cell fiber outgrowth assay of supernatants and recombinant BDNF analyzed in D. *Significantly different from EGFP with p < 0.01. Data in C and E are from >100 cells per experiment and from more than two independent preparations for each condition. Error bars represent SEs.

Article Snippet: Recombinant human NTs were purchased from Alamone Labs. Monensin was obtained from Sigma.

Techniques: Transfection, Western Blot, Incubation, Recombinant, Expressing

Journal: The Journal of Neuroscience

Article Title: Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

doi: 10.1523/JNEUROSCI.1776-05.2005

Figure Lengend Snippet: Neutralization of pH in secretory granules by monensin before depolarization speeds up the release of BDNF. Hippocampal neurons were transfected with GFP-tagged NTs at 8 DIV and used for secretion 2 d after transfection A, BDNF-GFP-expressing neuron. B, Time series of the marked box in A at higher magnification. C, Analysis of fluorescence intensity of regions marked in B. Note the pronounced increase in fluorescence intensity after application of monensin (4 μm), which neutralizes pH in previously acidic granules. The letters a- e refer to the pictures shown in B. Note the faster time course of BDNF secretion (compare Fig. 11) and the lack of GFP unquenching after K+-induced depolarization. rel., Relative; stimul., stimulation. Error bars represent SEs.

Article Snippet: Recombinant human NTs were purchased from Alamone Labs. Monensin was obtained from Sigma.

Techniques: Neutralization, Transfection, Expressing, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

doi: 10.1523/JNEUROSCI.1776-05.2005

Figure Lengend Snippet: Average time course of NT secretion after neutralization of intragranular pH with monensin. A-D, Hippocampal neurons expressing the different GFP-tagged NTs were treated as in Figure 9. Monensin (4 μm)-induced fluorescence increase and acceleration of the time course of release during subsequent depolarization was observed for all NTs (compare Fig. 11). The τ values represent the results of monoexponential fit curves for n ≥ 4 cells for each NT. E, Normalized average depolarization induced (50 mm K+) fluorescence decrease for indicated NTs after preincubation with monensin and for FM4-64 destaining of synaptic vesicles, respectively. The vertical line indicates start of depolarization. Average over n ≥ 4 cells for each condition. F, The boxed area in E at an expanded time scale. Note the similar half decay times (gray circles) for all NTs under these conditions but the still markedly faster speed of FM4-64 release from synaptic vesicles. Stimul., Stimulation. Error bars represent SEs.

Article Snippet: Recombinant human NTs were purchased from Alamone Labs. Monensin was obtained from Sigma.

Techniques: Neutralization, Expressing, Fluorescence