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( A ) A focal mosaic of PCDH19 expression was created by injecting an <t>AAV</t> expressing EGFP-Cre <t>recombinase</t> into the right visual cortex (V1) of the PCDH19flox/flox mouse. The opposite, non-injected hemisphere was used as the internal control. ( B ) PCDH19 mosaic mice experienced a transient period of unexpected mortality. Data collected from 70 mice injected at P1 (blue) differs significantly from that of control littermates (N = 48 mice, black; Mantel-Cox test p < 0.01). Yellow area indicates the period of adolescence in mice. ( C ) PCDH19 mice have a normal diurnal alternation of resting phases during the day (light on) and of high locomotor activity during the night (light off, shaded area; darkness from 7 PM till 7 AM). Bars represent the speed averaged in 5 min bins measured in three control and three PCDH19 mice in a 48-h period. Recordings start at 12 AM. ( D ) Density maps showing the arena occupation during a 2-h period of the active phase. ( E ) PCDH19 mice are hyperactive during the night phase. ( F ) PCDH19 mice spend less time than controls in resting states that can be ascribed to sleep (N = 8 for both control and PCDH19 mice; data pooled from 36 and 38 days of video recording). Significant differences are indicated with asterisks (** p < 0.02, Mann–Whitney test).
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( A ) A focal mosaic of PCDH19 expression was created by injecting an <t>AAV</t> expressing EGFP-Cre <t>recombinase</t> into the right visual cortex (V1) of the PCDH19flox/flox mouse. The opposite, non-injected hemisphere was used as the internal control. ( B ) PCDH19 mosaic mice experienced a transient period of unexpected mortality. Data collected from 70 mice injected at P1 (blue) differs significantly from that of control littermates (N = 48 mice, black; Mantel-Cox test p < 0.01). Yellow area indicates the period of adolescence in mice. ( C ) PCDH19 mice have a normal diurnal alternation of resting phases during the day (light on) and of high locomotor activity during the night (light off, shaded area; darkness from 7 PM till 7 AM). Bars represent the speed averaged in 5 min bins measured in three control and three PCDH19 mice in a 48-h period. Recordings start at 12 AM. ( D ) Density maps showing the arena occupation during a 2-h period of the active phase. ( E ) PCDH19 mice are hyperactive during the night phase. ( F ) PCDH19 mice spend less time than controls in resting states that can be ascribed to sleep (N = 8 for both control and PCDH19 mice; data pooled from 36 and 38 days of video recording). Significant differences are indicated with asterisks (** p < 0.02, Mann–Whitney test).
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Figure 3. TeNT-LC or BoNT/B overexpression does not promote axon growth in adult DRG neurons (A) Immunoblot of VAMP2 and Tuj1 in L3–L5 DRG extracts 3 weeks after <t>AAV-TeNT-LC-P2A-mCherry</t> or AAV-tdTomato injection into wild-type mice. (B) Quantification of (A). Values are plotted as mean ± SEM; ***p < 0.001 by Student’s t test; n = 3 independent experiments. (C) Representative fluorescence images of Tuj1 (cyan) and TeNT-LC-P2A-mCherry/tdTomato (red) immunolabeled DRG neurons 3 weeks after AAV adminis- tration and plated for 15–16 h. Scale bar, 100 mm. (D and E) Length of the longest axon (D) and branching frequency (E) of (C). Values are plotted as mean ± SEM; n = 3–4 independent experiments with 145 tdTomato+ neurons, 133 TeNT-LC-P2A-mCherry+ neurons. (F) Immunoblot of VAMP2 and Tuj1 in L3–L5 DRG extracts 3 weeks after AAV-Cre-GFP injection into iBOT and wild-type control mice. (G) Quantification of (F). Values are plotted as mean ± SEM;*p < 0.05 by Student’s t test. n = 3 independent experiments. (H) Representative fluorescence images of Tuj1 (red) and Cre-GFP (cyan) immunolabeled DRG neurons 3 weeks after AAV-Cre-GFP administration and plated for 15–16 h. Scale bar, 100 mm. (I and J) Length of the longest axon (I) and branching frequency (J) of (H). Values are plotted as mean ± SEM; n = 3 independent experiments with 100 Cre-GFP+
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( A ) A focal mosaic of PCDH19 expression was created by injecting an AAV expressing EGFP-Cre recombinase into the right visual cortex (V1) of the PCDH19flox/flox mouse. The opposite, non-injected hemisphere was used as the internal control. ( B ) PCDH19 mosaic mice experienced a transient period of unexpected mortality. Data collected from 70 mice injected at P1 (blue) differs significantly from that of control littermates (N = 48 mice, black; Mantel-Cox test p < 0.01). Yellow area indicates the period of adolescence in mice. ( C ) PCDH19 mice have a normal diurnal alternation of resting phases during the day (light on) and of high locomotor activity during the night (light off, shaded area; darkness from 7 PM till 7 AM). Bars represent the speed averaged in 5 min bins measured in three control and three PCDH19 mice in a 48-h period. Recordings start at 12 AM. ( D ) Density maps showing the arena occupation during a 2-h period of the active phase. ( E ) PCDH19 mice are hyperactive during the night phase. ( F ) PCDH19 mice spend less time than controls in resting states that can be ascribed to sleep (N = 8 for both control and PCDH19 mice; data pooled from 36 and 38 days of video recording). Significant differences are indicated with asterisks (** p < 0.02, Mann–Whitney test).

Journal: Cells

Article Title: Perturbation of Cortical Excitability in a Conditional Model of PCDH19 Disorder

doi: 10.3390/cells11121939

Figure Lengend Snippet: ( A ) A focal mosaic of PCDH19 expression was created by injecting an AAV expressing EGFP-Cre recombinase into the right visual cortex (V1) of the PCDH19flox/flox mouse. The opposite, non-injected hemisphere was used as the internal control. ( B ) PCDH19 mosaic mice experienced a transient period of unexpected mortality. Data collected from 70 mice injected at P1 (blue) differs significantly from that of control littermates (N = 48 mice, black; Mantel-Cox test p < 0.01). Yellow area indicates the period of adolescence in mice. ( C ) PCDH19 mice have a normal diurnal alternation of resting phases during the day (light on) and of high locomotor activity during the night (light off, shaded area; darkness from 7 PM till 7 AM). Bars represent the speed averaged in 5 min bins measured in three control and three PCDH19 mice in a 48-h period. Recordings start at 12 AM. ( D ) Density maps showing the arena occupation during a 2-h period of the active phase. ( E ) PCDH19 mice are hyperactive during the night phase. ( F ) PCDH19 mice spend less time than controls in resting states that can be ascribed to sleep (N = 8 for both control and PCDH19 mice; data pooled from 36 and 38 days of video recording). Significant differences are indicated with asterisks (** p < 0.02, Mann–Whitney test).

Article Snippet: Mosaic PCDH19 expression was induced in the occipital cortex of PCDH19 conditional knockout mice by focal injection of an AAV expressing EGFP-Cre recombinase (Addgene #105545-AAV1; pAAV.CMV.HI.eGFP-Cre.WPRE.SV40).

Techniques: Expressing, Injection, Control, Activity Assay, MANN-WHITNEY

In vivo 2-photon calcium imaging in PCDH19 mosaic brain. ( A ) Example field of view with Cre-negative red neurons (top grey square; expressing jRGECO1a only) and Cre positive (PCDH19 knockout) green neurons (bottom square) that express jRGECO1a in the cytoplasm and EGFP-Cre recombinase in the nucleus. ( B ) Calcium transients (green trace) measured over the entire field of view. The vertical axis displays the number of pixels affected by calcium activity over time (see Supplemental Information). The black trace shows the LFP recorded from the same hemisphere with an electrode positioned just outside of the imaged field at a depth of 250 µm. The inset shows magnified traces in correspondence of the black bar. Notice how Ca 2+ transients are phase locked to the USs. ( C ) Frequency of calcium transients in red and green cells of mosaic mice and controls. Each dot represents the average value for each active neuron. Significant differences are indicated with asterisks (*** p < 0.0005, ** p < 0.005; Mann–Whitney test). Transient frequency is significantly increased in mosaics compared to control mice both for Cre+ (PCDH19 KO) neurons and for Cre- neurons. ( D ) Box plots of the percentage of Ca 2+ transients occurring within USs, demonstrating a significant reduction in synchronization with the SWA oscillation in mosaic mice compared to control mice (* p < 0.02; Mann–Whitney test, whiskers indicate data range). Each dot represents the value obtained by all neurons in the imaging field of PCDH19 mosaic mice. The filled magenta dots represent the values obtained from mosaic animals with a hyperexcitable (>5 β oscillations per hour) phenotype, the blue dots from all other mosaics.

Journal: Cells

Article Title: Perturbation of Cortical Excitability in a Conditional Model of PCDH19 Disorder

doi: 10.3390/cells11121939

Figure Lengend Snippet: In vivo 2-photon calcium imaging in PCDH19 mosaic brain. ( A ) Example field of view with Cre-negative red neurons (top grey square; expressing jRGECO1a only) and Cre positive (PCDH19 knockout) green neurons (bottom square) that express jRGECO1a in the cytoplasm and EGFP-Cre recombinase in the nucleus. ( B ) Calcium transients (green trace) measured over the entire field of view. The vertical axis displays the number of pixels affected by calcium activity over time (see Supplemental Information). The black trace shows the LFP recorded from the same hemisphere with an electrode positioned just outside of the imaged field at a depth of 250 µm. The inset shows magnified traces in correspondence of the black bar. Notice how Ca 2+ transients are phase locked to the USs. ( C ) Frequency of calcium transients in red and green cells of mosaic mice and controls. Each dot represents the average value for each active neuron. Significant differences are indicated with asterisks (*** p < 0.0005, ** p < 0.005; Mann–Whitney test). Transient frequency is significantly increased in mosaics compared to control mice both for Cre+ (PCDH19 KO) neurons and for Cre- neurons. ( D ) Box plots of the percentage of Ca 2+ transients occurring within USs, demonstrating a significant reduction in synchronization with the SWA oscillation in mosaic mice compared to control mice (* p < 0.02; Mann–Whitney test, whiskers indicate data range). Each dot represents the value obtained by all neurons in the imaging field of PCDH19 mosaic mice. The filled magenta dots represent the values obtained from mosaic animals with a hyperexcitable (>5 β oscillations per hour) phenotype, the blue dots from all other mosaics.

Article Snippet: Mosaic PCDH19 expression was induced in the occipital cortex of PCDH19 conditional knockout mice by focal injection of an AAV expressing EGFP-Cre recombinase (Addgene #105545-AAV1; pAAV.CMV.HI.eGFP-Cre.WPRE.SV40).

Techniques: In Vivo, Imaging, Expressing, Knock-Out, Activity Assay, MANN-WHITNEY, Control

Figure 3. TeNT-LC or BoNT/B overexpression does not promote axon growth in adult DRG neurons (A) Immunoblot of VAMP2 and Tuj1 in L3–L5 DRG extracts 3 weeks after AAV-TeNT-LC-P2A-mCherry or AAV-tdTomato injection into wild-type mice. (B) Quantification of (A). Values are plotted as mean ± SEM; ***p < 0.001 by Student’s t test; n = 3 independent experiments. (C) Representative fluorescence images of Tuj1 (cyan) and TeNT-LC-P2A-mCherry/tdTomato (red) immunolabeled DRG neurons 3 weeks after AAV adminis- tration and plated for 15–16 h. Scale bar, 100 mm. (D and E) Length of the longest axon (D) and branching frequency (E) of (C). Values are plotted as mean ± SEM; n = 3–4 independent experiments with 145 tdTomato+ neurons, 133 TeNT-LC-P2A-mCherry+ neurons. (F) Immunoblot of VAMP2 and Tuj1 in L3–L5 DRG extracts 3 weeks after AAV-Cre-GFP injection into iBOT and wild-type control mice. (G) Quantification of (F). Values are plotted as mean ± SEM;*p < 0.05 by Student’s t test. n = 3 independent experiments. (H) Representative fluorescence images of Tuj1 (red) and Cre-GFP (cyan) immunolabeled DRG neurons 3 weeks after AAV-Cre-GFP administration and plated for 15–16 h. Scale bar, 100 mm. (I and J) Length of the longest axon (I) and branching frequency (J) of (H). Values are plotted as mean ± SEM; n = 3 independent experiments with 100 Cre-GFP+

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: Figure 3. TeNT-LC or BoNT/B overexpression does not promote axon growth in adult DRG neurons (A) Immunoblot of VAMP2 and Tuj1 in L3–L5 DRG extracts 3 weeks after AAV-TeNT-LC-P2A-mCherry or AAV-tdTomato injection into wild-type mice. (B) Quantification of (A). Values are plotted as mean ± SEM; ***p < 0.001 by Student’s t test; n = 3 independent experiments. (C) Representative fluorescence images of Tuj1 (cyan) and TeNT-LC-P2A-mCherry/tdTomato (red) immunolabeled DRG neurons 3 weeks after AAV adminis- tration and plated for 15–16 h. Scale bar, 100 mm. (D and E) Length of the longest axon (D) and branching frequency (E) of (C). Values are plotted as mean ± SEM; n = 3–4 independent experiments with 145 tdTomato+ neurons, 133 TeNT-LC-P2A-mCherry+ neurons. (F) Immunoblot of VAMP2 and Tuj1 in L3–L5 DRG extracts 3 weeks after AAV-Cre-GFP injection into iBOT and wild-type control mice. (G) Quantification of (F). Values are plotted as mean ± SEM;*p < 0.05 by Student’s t test. n = 3 independent experiments. (H) Representative fluorescence images of Tuj1 (red) and Cre-GFP (cyan) immunolabeled DRG neurons 3 weeks after AAV-Cre-GFP administration and plated for 15–16 h. Scale bar, 100 mm. (I and J) Length of the longest axon (I) and branching frequency (J) of (H). Values are plotted as mean ± SEM; n = 3 independent experiments with 100 Cre-GFP+

Article Snippet: Reagent or resource Source Identifier Antibodies Rabbit anti-Tubulin b 3 (TUBB3) Sigma-Aldrich T2200; RRID: AB_262133 Chicken anti-GFP Abcam ab13970; RRID: AB_300798 Mouse anti-tubulin b 3 (Tuj-1) Biolegend 801201; RRID: AB_2313773 Mouse anti-VAMP2 Synaptic Systems 104211; RRID: AB_887811 Rabbit anti-Munc13-1 Synaptic Systems 126103; RRID: AB_887733 Rabbit anti-RIM1 Synaptic Systems 140103; RRID: AB_887774 Rabbit anti-RIM2 Synaptic Systems 140 103; RRID: AB_88776 Rabbit anti-Glial Fibrillary Acidic Protein (GFAP) DAKO Z0334; RRID: AB_10013382 Rabbit anti-RFP Rockland 600-401-379; RRID: AB_2209751 Mouse anti-DDK (FLAG) Origene TA50011-100; RRID: AB_2622345 Guinea Pig anti-NeuN Merck Millipore ABN90P; RRID: AB_2341095 Donkey anti-chicken IgY IgG H+L Alexa Fluor 488 Jackson Immunoresearch 703-545-155; RRID: AB_2340375 Donkey anti-rabbit IgG H+L Alexa Fluor 594 Jackson Immunoresearch 711-586-152; RRID: AB_2340622 Donkey anti-guinea pig IgG H+L Alexa Fluor 647 Jackson Immunoresearch 706-605-148; RRID: AB_2340476 Goat anti-rabbit IgG H+L Alexa Fluor 568 Thermo Fisher A-11036; RRID: AB_10563566 Goat Anti-chicken IgY H&L Alexa Fluor 488 Abcam ab150169; RRID: AB_2636803 Goat anti-rabbit IgG H&L Alexa Fluor 647 Abcam ab150079; RRID: AB_2722623 Goat anti-Mouse IgG H+L Alexa Fluor 647 Thermo Fisher A-21235; RRID: AB_2535804350 Goat anti-Mouse IgG H+ Alexa Fluor 594 Thermo Fisher A-11032; RRID: AB_2534091 Bacterial and virus strains AAV5.CAG-tdTomato (codon diversified) A gift from Edward Boyden Addgene #59462-AAV5 AAV1.CMV.PI.EGFP.WPRE.bGH A gift from James M. Wilson Addgene #105530-AAV1 AAV1.CMV.HI.eGFP-Cre.WPRE.SV40 A gift from James M. Wilson Addgene #105545-AAV1 AAV1.CAG.Cre.mCherry SignaGen SL101117 AAV5.hSyn.hM4D(Gi)-mCherry A gift from Bryan Roth Addgene #50475-AAV5 AAV1.CMV-TeNT-LC-P2A-mCherry Sirion Biotech N/A Chemicals, peptides, and recombinant proteins HBSS GIBCO 14025-053 HEPES GIBCO 15630-56 DMEM/F-12, HEPES Life Technologies 11330032 Fetal bovine serum Thermo Scientific Cat#10500064 Penicillin/Steptomycin (PenStrep) antibiotics GIBCO 15140122 Collagenase IV Sigma C1889 Trypsin Worthington LS003703 DNase Worthington LS002007 Neurobasal medium GIBCO 12349-015 B-27 supplement GIBCO 17504-044 L-glutamine GIBCO 25030-024 Bovine serum albumin Sigma A3294 Horse serum Pan Biotech P30-0712 Poly-L-lysine Sigma P2636 Laminin Roche 11243217001 Phosphate buffer solution 0.1 M Sigma P5244 (Continued on next page) e1 Neuron 110, 51–69.e1–e7, January 5, 2022

Techniques: Over Expression, Western Blot, Injection, Immunolabeling, Control