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. 2006 Sep 7;25(18):4361–4371. doi: 10.1038/sj.emboj.7601318

Figure 3.

Figure 3

Tetanus induces prS remodeling in cultured hippocampal neurons to enhance transmitter release. (A) Example of an axon expressing VAMP2-GFP fusion protein. Scale bar 10 μm. (B, C) Examples of punctated (B) and diffused (C) fluorescence from the axon expressing VAMP2-GFP fusion protein shown in (A). Scale bar 2 μm. (D) Examples of activity-dependent staining of FM4-64 before and after tetanus in cultures transfected with VAMP2-GFP. Tetanus increased number of yellow puncta (FM4-64 staining in VAMP2-GFP expressing axon) as well as red puncta (FM4-64 staining in nontransfected axons). Tetanus also increased releasable fluorescence in btns from both transfected and nontransfected axons. D-AP5 (40 μM) blocked tetanus-induced increase in btn number and releasable fluorescence (one-way ANOVA followed by LSD). Scale bar 10 μm. (E) Insets show a VAMP2-GFP punctum that becomes recycling after tetanus. Scale bar 1 μm. (F) An example of fusion of diffused VAMP2-GFP to become a recycling btn. The dashed line shows the diffused fluorescence. The arrow shows the new btn. Scale bar 1 μm. (G) An example of budding of new VAMP2-GFP btns from a pre-existing VAMP2-GFP btn. The yellow arrow shows the original btn whereas the white arrow shows the new btns. Scale bar 1 μm. (H) A pie chart representing percentage contribution of (1) conversion of nonrecycling pre-existing btns to recycling btns (74%), (2) fusion of nonsynaptic VAMP2-GFP (14.3%) and (3) budding from pre-existing recycling btns (11.7%) in tetanus-induced increase in VAMP2-GFP recycling btn number calculated from 15 samples. (I) Number of FM4-64 stained btns per 30 μm of VAMP2-GFP expressing axons in control (8), tetanus (15), L-NMA paired with tetanus (12), KT5823 paired with tetanus (11), KN-93 paired with tetanus (8) and KN-92 paired with tetanus (8) groups before (filled bar) and 30 min after (empty bar) the tetanus. The empty bar in the control group represents parallel measurement of number of FM4-64 positive VAMP2-GFP puncta at 30 min without tetanus. L-NMA, KT5823, KN-93 but not KN-92 blocked tetanus-induced increase in FM4-64 recycling btn number. In L-NMA, KT5823, KN-93 but not KN-92 groups, we neither observed fusion of nonsynaptic VAMP2-GFP nor budding from recycling VAMP2-GFP btns. *P<0.001 compared to pretetanus value.