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. 2020 May 28;23(6):101203. doi: 10.1016/j.isci.2020.101203

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© 2020 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

PTPσ Deletion Reduces Vesicle Localization in Excitatory Presynaptic Boutons

(A) Representative traces of AMPAR-EPSCs evoked by single 2-s pulse of 0.5 M sucrose delivered at a 1-min interval, recorded from hippocampal cultured neurons derived from PTPσ^f/f mice infected with lentiviruses expressing inactive (ΔCre) or active (Cre) Cre recombinase.

(B and C) Bar graphs showing charge transfer (B) and peak amplitudes (C) of sucrose-evoked EPSCs, estimated as the synaptic charge transfer integrated over 30 s. Recordings were performed in the presence of 1 μM tetrodoxin and 50 μM picrotoxin. Data are means ± SEMs (n denotes number of analyzed neurons; ΔCre, 26 and Cre, 29; ^∗p < 0.05; ^∗∗∗p < 0.001; unpaired t test).

(D) Representative images of cultured neurons (DIV10) derived from PTPσ^f/f mice infected with lentiviruses expressing ΔCre or Cre at DIV3 and transfected with VGLUT1-mVenus (green) at DIV8. Anti-Bassoon (red) was used to mark the presynaptic active zone. Scale bar: 10 μm.

(E) Quantification of synaptic vesicle diffusion from images in (D), determined by measuring the average length of the major axis of VGLUT1-mVenus fluorescence in transfected axons. Data are means ± SEMs (n denotes the number of analyzed neurons; ΔCre, n = 19; Cre, n = 18; Cre+/PTPσ WT, n = 15; Cre+/PTPσ C1157S, n = 15; and Cre+/PTPσ ΔD2, n = 17; ^∗∗∗∗p < 0.0001; ANOVA with a non-parametric Kruskal-Wallis test).

(F) Quantification of VGLUT1-mVenus fluorescence enrichment at presynaptic active zone for the images in (D). Data are means ± SEMs (n denotes the number of analyzed neurons; ΔCre, n = 19; Cre, n = 18; Cre+/PTPσ WT, n = 15; Cre+/PTPσ C1157S, n = 15; and Cre+/PTPσ ΔD2, n = 17; ANOVA with a non-parametric Kruskal-Wallis test).

(G) Representative images of cultured neurons (DIV10) derived from PTPσ^f/f mice infected with lentiviruses expressing ΔCre or Cre at DIV3 and transfected with EGFP (green) at DIV8. Anti-Bassoon (blue) was used to mark the presynaptic active zone. Scale bar: 10 μm.

(H) Quantification of synaptic vesicle diffusion from images in (G), determined by measuring the average length of the major axis of VGLUT1 fluorescence in transfected axons. Data are means ± SEMs (n denotes the number of analyzed neurons; ΔCre, n = 14; Cre, n = 12; Cre+/PTPσ WT, n = 13; Cre+/PTPσ C1157S, n = 15; and Cre+/PTPσ ΔD2, n = 13; ^∗∗∗∗p < 0.0001; ANOVA with a non-parametric Kruskal-Wallis test).

(I) Quantification of VGLUT1 fluorescence enrichment at presynaptic active zone for the images in (G). Data are means ± SEMs (n denotes the number of analyzed neurons; ΔCre, n = 14; Cre, n = 12; Cre+/PTPσ WT, n = 13; Cre+/PTPσ C1157S, n = 15; and Cre+/PTPσ ΔD2, n = 13; ANOVA with a non-parametric Kruskal-Wallis test).