Results: 4

1.
Fig. 2.

Fig. 2. From: Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons.

ShhN-induced presynaptic terminals internalize more endocytic markers. Representative images of neurons that have endocytosed a fixable FM dye (A), an antibody to the luminal domain of Synaptotagmin1 (Syt1; C), or an antibody to the luminal domain of VGAT (E). The ShhN-treated neurons exhibit an increased number of FM dye- (B), Syt1- (D) and VGAT- (F) antibody-containing synapses. Scale bars: 20 µm. n = 60 neurons for FM dye uptake, n = 30 cells for Syt1 and VGAT antibody uptake. Additional images and analyses are shown in supplementary material Fig. S5.

Nicholas Mitchell, et al. J Cell Sci. 2012 September 15;125(18):4207-4213.
2.
Fig. 4.

Fig. 4. From: Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons.

Shh activity enhances neuronal excitability and presynaptic neurotransmitter release. (A) Neurons exhibiting resting membrane potential between −60 and −70 mV were exposed to current steps of 20 pA increments to depolarize and produce APs. The ShhN-treated neurons fire APs more readily than controls. (B) AP frequency at three different current steps. n = 13 cells for controls, n = 12 for ShhN-treated neurons. *P<0.05. (C) The accumulation of APs (averages used), as current intensity increases. (D) Sample traces of mEPSC recordings from control and ShhN-treated neurons. Histograms depict mean frequency (E) and amplitude (F) of mEPSC events. n = 7 for controls, n = 9 for ShhN-treated neurons. ***P<0.001.

Nicholas Mitchell, et al. J Cell Sci. 2012 September 15;125(18):4207-4213.
3.
Fig. 3.

Fig. 3. From: Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons.

Shh activity enlarges presynaptic terminals and synaptic vesicles. Representative electron micrographs of synapses from control (AC) and ShhN-treated neurons (DG), both of which form synapses on spines (sp) and dendrite shafts (de). Note the enlargement of presynaptic terminals (pre) and the synaptic vesicles within the terminals, following ShhN treatment. (D) A presynaptic terminal forming synapses on two spines. (E) A degenerating process (asterisk) above the presynaptic terminal that contains autophagosomes and multivesicular bodies. (G) A very large terminal forming a synapse with a cell soma (so). In addition to enlarged synaptic vesicles, this terminal contains tubulovesicular structures associated with endosomes (en). Dark organelles in the presynaptic terminals in F and G are mitochondria. Scale bar: 100 nm (A–G). (H) Distribution and average size of presynaptic terminals. n = 229 terminals in both groups. (I) Distribution and average size of synaptic vesicles. ***P<0.001. Additional analyses and examples of ShhN-elicited mitochondria-enriched terminals are shown in supplementary material Fig. S6.

Nicholas Mitchell, et al. J Cell Sci. 2012 September 15;125(18):4207-4213.
4.
Fig. 1.

Fig. 1. From: Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons.

Shh signaling induces formation of presynaptic terminals. (A) Hippocampal neurons treated with ShhN were labeled for the indicated presynaptic markers (a, c, e). Scale bars: 20 µm. Histograms show quantification of presynaptic puncta (b, d, f). n = 80 neurons for Synapsin1; n = 60 neurons for Bassoon and ZnT3. SAG (100 nM), a Shh agonist; Cyclopamine (10 µM), a Shh antagonist. (B) Neurons were labeled for (a) VGlut and PSD95, or (b) VGAT and Gephyrin (Gphn). Scale bars: 5 µm. In the ShhN-treated neurons, some of the presynaptic VGlut puncta do not pair with visible PSD95 puncta (white arrows in a). Likewise, the ShhN neurons have more VGAT puncta free of apposing Gphn puncta (white arrows in b). The proportion of free VGlut puncta (c) and free VGAT puncta (d) are greater in the ShhN neurons. n ≧200 total puncta in each group. (C) Immunoblots showing that the expression level of Bassoon, but not Synapsin1 or Synaptophysin, is increased in response to Shh activity. Purm (purmorphamine; 3.6 µM) is another Shh agonist. A second independent set of blots yielded similar results (not shown). (D) Representative images of synapses co-expressing presynaptic Synaptophysin::EGFP (Syp::EGFP) (a) or postsynaptic PSD95::EGFP (b) with a control vector or SmoA. Scale bars: 5 µm. Cumulative distribution analysis shows that SmoA expression elicits an increased size of Syp::EGFP synapses and this increase is more evident in ∼21 div neurons (white and black squares) than in ∼14 div neurons (white and black triangles). By contrast, the size of PSD95::EGFP synapses is not significantly altered by SmoA, in either age group. n = 346–506 puncta. All data are mean ± s.e.m. ***P<0.001, **P<0.01, *P<0.05, Student's t-test. Additional images and analyses are shown in supplementary material Figs S2–S4.

Nicholas Mitchell, et al. J Cell Sci. 2012 September 15;125(18):4207-4213.

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