The Journal of Neuroscience, August 17, 2005, ():
Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins
J. Neurosci. Brigadski et al.
25: 7601
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Fig.1: Differential distal and proximal vesicular targeting of overexpressed untagged NTs. Hippocampal neurons were transfected at 8 DIV with the different untagged NTs and immunostained at 10 DIV with antibodies directed against the respective heterologously expressed NT. Distal vesicular (A) and proximal expressers (B) were observed for all untagged NTs, similar to the pattern in cells expressing the GFP tagged versions. C: The percentage of distal vesicular expressers of BDNF (72.9 ± 1.1%) and NT-4 (47.8 ± 2.6%) was quantified and yielded significantly less (p<10-5) distal expressers in NT-4 expressing cells.
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Supplementary Fig.2: Localization of endogenous NTs. Untransfected hippocampal neurons were processed for immunocytochemistry at 10 DIV to localize endogenously expressed NTs. In many cells the endogenous NTs were below the levels of detection. A-D give examples of distal expressers for all NTs, as indicated by the punctate staining in the processes (Lower panels show boxed areas at higher magnification). The typical staining pattern of proximal expressers which is characterized by the absence of NT vesicles in dendrites and by the presence of faint dendritic fluorescence (compare Fig.2 for GFP tagged NTs), could not be distinguished from unspecific background fluorescence (data not shown).
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Supplementary Fig.3: Similar targeting of all GFP tagged NTs in PC12 cells. PC12 cells were differentiated with 100 ng/ml NGF, and were transfected with the different GFP tagged NTs at 7 DIV. GFP fluorescence was monitored 2 days posttransfection. A: Typical examples of distal vesicular expression of all NTs. B: Examples of PC12 cells showing the proximal expression pattern. C: Quantification of distal expression pattern of all NTs in differentiated PC12. All NTs showed distal vesicular targeting in 70-90 % of transfected cells (n>50 cells for each NT). Thus the clonal PC12 cells – when differentiated – promote preferential distal vesiculartargeting of all NTs. D: Undifferentiated PC12 cells – when transfected with GFP tagged NTs – displayed exclusive proximal expression for all NTs (shown here for NT-3).
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Supplementary Fig.4: NTs are targeted to active synapses of hippocampal neurons. Hippocampal neurons were transfected at 8 DIV with the respective GFP tagged NT and active presynaptic terminals were stained with FM 4-64 at 10 DIV. In A-E: GFP fluorescence signals (left), red fluorescent FM 4-64 (middle) and a merged picture of both fluorescence channels (right) are shown for the same field of view. A: NT-3-GFP expressing neuron at low magnification. White box is shown in B at higher magnification. Note the apposition of NT-3-GFP vesicle containing dendrites with active presynaptic terminals (arrowheads) C-E: Examples of FM 4-64 staining for the other NTs at high magnification, showing dendritic delivery of all four NTs to active synapses in hippocampal neurons.
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Supplementary Fig.5: Specificity control for the anti-NT-4 antibody. Untransfected hippocampal neurons were processed at 10DIV for immunocytochemistry as described in Materials and Methods. A: Incubation with primary anti-NT-4 antibody (1:200, 24h, RT) and subsequent incubation (1h, RT) with the secondary antibody (Alexa Red coupled anti-sheep, 1:500). Note the granular staining for NT-4 in the cell body (arrow heads) and in the neurites (arrows). B: Negative control: same procedure as in A, but a 25fold molar excess of human recombinant NT-4 (i.e 25 µg/ml) was added to the final primary NT-4 antibody dilution 1h prior to incubation with the cells, in order to saturate all NT-4 specific antibody binding sites with the excess NT-4. Note the absence of granular staining in the soma (arrowhead) or in the neurites. Pictures in A and B show confocal images that were taken with identical laser intensities and exposure times (3s).
