Journal: Neuron

Article Title: An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury

doi: 10.1016/j.neuron.2021.10.007

Figure Lengend Snippet: RIM1/2 deletion promotes axon growth in adult DRG neurons (A) Normalized expression values of RIMS1 and RIMS2 in DRG at E12.5, E17.5, and adult from 6 to 36 h in culture. ∗∗∗ p < 0.001 6 versus 24 or 36 h, ∗ p < 0.05 6 versus 24 or 36 h in culture by one-way ANOVA followed by Tukey’s post hoc test; n = 3 technical replicates per condition. (B) Immunoblot of RIM1, RIM2, and Tuj1 in L3–L5 DRG extracts after 6 or 36 h in culture. (C and D) Quantification of RIM1 (C) and RIM2 (D) in (B). Values are plotted as mean ± SEM; ∗∗ p < 0.01, #p < 0.10 by Student’s t test; n = 3 independent experiments. (E) Immunoblot of RIM1 and Tuj1 in L3–L5 DRG extracts electroporated with Rim1α-GFP or GFP (control)-expressing plasmids and cultured for 24 h. (F) Quantification of (E). Values are plotted as mean ± SEM; ∗∗ p < 0.01 by Student’s t test. n = 3 independent experiments. (G) Representative fluorescence images of naive DRG neurons electroporated with RIM1α-GFP or GFP-expressing plasmids and cultured for 24 h. Scale bar, 100 μm. (H and I) Length of the longest axon (H) and branching frequency (I) of (G). Values are plotted as mean ± SEM; ∗∗ p < 0.01, ∗ p < 0.05 control GFP versus RIM1α-GFP by Student’s t test; n = 3 independent experiments with 103 control GFP + neurons, 141 RIM1α-GFP + neurons. (J) Representative images of cultured DRG neurons after AAV-Cre-GFP administration into RIM1 fl/fl RIM2 fl/fl mice. Cyan arrowhead points to Cre-GFP + neuron and white arrowhead points to Cre-GFP – neuron. Scale bar, 15 μm. (K) Quantification of (J). Values are plotted as mean ± SEM; ∗∗ p < 0.01 by Student’s t test; n = 3 independent experiments, 106 GFP + neurons, and 98 Cre-GFP + neurons. (L) Representative fluorescence images of Tuj1 (red) and GFP/Cre-GFP (cyan)-immunolabeled DRG neurons 3 weeks after AAV-GFP or AAV-Cre-GFP administration into RIM1 fl/fl RIM2 fl/fl mice and plated for 15–16 h. The AAV-Cre-GFP image shows the same neurons as in (J). Scale bar, 100 μm. (M and N) Length of the longest axon (M) and branching frequency (N) of (L). Values are plotted as mean ± SEM; ∗∗∗ p < 0.001, ∗∗ p < 0.01 by Student’s t test. n = 3 independent experiments, 108 GFP + neurons, 101 Cre-GFP+ neurons.

Article Snippet: The following primary antibodies were used: anti-VAMP2 (1:1000, Synaptic Systems #104 211, mouse monoclonal), anti-Munc13-1 (1:2000, Synaptic Systems #126 103, rabbit polyclonal), anti-RIM1 (1:500; Synaptic Systems #140 003, rabbit polyclonal), anti-RIM2 (1:500; Synaptic Systems #140 103, rabbit polyclonal), anti-tuj1 (1:10000, Sigma #T2200, rabbit polyclonal).

Techniques: Expressing, Western Blot, Cell Culture, Fluorescence, Immunolabeling

Journal: Neuron

Article Title: An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury

doi: 10.1016/j.neuron.2021.10.007

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: anti-VAMP2 (1:1000, Synaptic Systems #104 211, mouse monoclonal), anti-Munc13-1 (1:2000, Synaptic Systems #126 103, rabbit polyclonal), anti-RIM1 (1:500; Synaptic Systems #140 003, rabbit polyclonal), anti-RIM2 (1:500; Synaptic Systems #140 103, rabbit polyclonal), anti-tuj1 (1:10000, Sigma #T2200, rabbit polyclonal).

Techniques: Recombinant, Protease Inhibitor, Software, Microscopy, Plasmid Preparation, Fluorescence, Incubation, Imaging

Journal: Frontiers in Neural Circuits

Article Title: Activity-Dependent Remodeling of Synaptic Protein Organization Revealed by High Throughput Analysis of STED Nanoscopy Images

doi: 10.3389/fncir.2020.00057

Figure Lengend Snippet: Primary and secondary antibodies used for STED imaging.

Article Snippet: Rabbit anti-RIM1/2 , Synaptic Systems , 140203 , 1 : 500 , AA 1 to 466 from rat Rim2 , Dani et al. ( ); Tang et al. ( ) .

Techniques: Imaging, Purification, Recombinant

Journal: Frontiers in Neural Circuits

Article Title: Activity-Dependent Remodeling of Synaptic Protein Organization Revealed by High Throughput Analysis of STED Nanoscopy Images

doi: 10.3389/fncir.2020.00057

Figure Lengend Snippet: Coupling properties of synaptic scaffold protein pairs at low neuronal activity measured with pySODA. (A) Schematic representation of the axial positions of the four synaptic scaffold proteins analyzed in this work. (B) Two examples of representative confocal and STED images of synaptic scaffold pairs. (C) Coupling probability and distance histograms measured for coupled cluster pairs of Bassoon - RIM1/2 (red, n = 16), PSD95 - Homer1c (yellow, n = 6), Bassoon - PSD95 (green, n = 18), Bassoon - Homer1c (blue, n = 17). Each 15 nm bin represents the average coupling probability calculated from the individual images of independent neurons with standard error. (D) Cumulative frequency plots of coupling distance (left) and coupling probability (right) for each synaptic element pair with standard error (shaded area). (C,D) n = number of neurons from 2 independent cultures. Statistical difference between scaffold protein pairs was assessed using a randomization test (see section Materials and Methods and ). Scale bar 250 nm.

Article Snippet: Rabbit anti-RIM1/2 , Synaptic Systems , 140203 , 1 : 500 , AA 1 to 466 from rat Rim2 , Dani et al. ( ); Tang et al. ( ) .

Techniques: Activity Assay

Journal: Frontiers in Neural Circuits

Article Title: Activity-Dependent Remodeling of Synaptic Protein Organization Revealed by High Throughput Analysis of STED Nanoscopy Images

doi: 10.3389/fncir.2020.00057

Figure Lengend Snippet: Activity-dependent re-organization of pre- and postsynaptic scaffolding protein pairs measured with the pySODA analysis framework. (A,E,I,M) Representative two-color STED images of synaptic protein pairs for the activity reducing high Mg 2+ /low Ca 2+ condition (left) or synaptic stimulation 0Mg 2+ /Gly/Bic (right) . Cumulative frequency graphs of the coupling distance (B,F,J,N) and of the coupling probability (C,G,K,O) measured for coupled protein pairs. (D,H,L,P) Histograms of the mean coupling probability per neuron at a given distance. Measurements were performed on two-color STED images of (A–D) the presynaptic protein pair Bassoon - RIM1/2 (High Mg 2+ / low Ca 2+ (gray): n = 16 and 0Mg 2+ /Gly/Bic (red): n = 21); (E–H) the postsynaptic protein pair PSD95 - Homer1c (high Mg 2+ / low Ca 2+ (gray): n = 9 and 0Mg 2+ /Gly/Bic (orange): n = 12); (I–L) the transsynaptic pair Bassoon - PSD95 (High Mg 2+ / low Ca 2+ (gray): n = 18 and 0Mg 2+ /Gly/Bic (green): n = 24) and (M–P) the transsynaptic pair Bassoon - Homer1c (High Mg 2+ / low Ca 2+ (gray): n = 17 and 0Mg 2+ /Gly/Bic (blue): n = 17). Shown are the means (plain lines) with standard error (shaded area). n = number of neurons from 3 independent cultures. Statistical difference was assessed using a randomization test (see section Materials and Methods and ). Exact p -values are reported in , with * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar 250 nm.

Article Snippet: Rabbit anti-RIM1/2 , Synaptic Systems , 140203 , 1 : 500 , AA 1 to 466 from rat Rim2 , Dani et al. ( ); Tang et al. ( ) .

Techniques: Activity Assay, Scaffolding