Journal: Translational psychiatry

Article Title: Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

doi: 10.1038/tp.2017.80

Figure Lengend Snippet: Figure 4. Signs of classical pro-inflammatory activation in Poly(I:C) hippocampal microglia. (a) Representative image of a sagittal slice (from a control animal) used for the analysis of the binding capacity with the TSPO-specific radioligand [18F]GE180. (b) Shows a representative image of a Nissl-stained sagittal slice (control animal) used to localize specific brain regions. (c) Shows that Poly(I:C) mice exhibit an increased binding potential to the TSPO in the hippocampus (n = 5 mice) with respect to controls (n = 6 mice). On the other hand, slices from Poly(I:C) mice treated with minocycline display a normalized binding potential in the latter region (n = 6 mice), significantly lower than untreated Poly(I:C) animals and comparable to controls. (d) Representative pictures illustrating increased Iba1 immunoreactivity in the proximity of the DG of Poly(I:C) mice as compared with controls and minocycline-treated mice. Pictures were taken at a 63-times magnification. (e) Iba1 immunoreactivity was increased in the dentate gyrus of the hippocampus (DG) of Poly(I:C) mice (n = 7) as compared with controls (n = 5). Poly (I:C) animals treated with minocycline (n = 4) displayed a normal Iba1 immunoreactivity. (f–h) Enzyme-linked immunosorbent assay (ELISA) measurement in whole hippocampal homogenates of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Only IL-6 was found to be significantly increased (h), while the levels of the other cytokines remained unchanged (f and g; Controls, n = 5; Poly(I:C), n = 5; Poly(I:C) treated with minocycline, n = 4). Error bars represent s.e.m. in all the panels. The data from the radioligand-binding assay were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The data from the immunoreactivity and ELISA were analyzed by one-way ANOVA followed by Newman–Keuls post hoc test. *Po0.05, **Po0.001. Cb, cerebellum; Cc, corpus callosum; ctrl., control animals; Ctx, cortex; Hip, hippocampus; Ob, olfactory bulbs; Poly, Poly(I:C) animals; Poly/Mino, Poly(I:C) animals treated with minocycline; Th, thalamus; TSPO, translocator protein.

Article Snippet: Complement component 4a (C4a) levels in hippocampal lysates were measured using the mouse C4a enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology, Wuhan, China), according to the manufacturer’s manual.

Techniques: Activation Assay, Control, Binding Assay, Staining, Enzyme-linked Immunosorbent Assay, Radio Ligand Binding Assay

Journal: Translational psychiatry

Article Title: Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

doi: 10.1038/tp.2017.80

Figure Lengend Snippet: Figure 5. Changes in the complement system in the hippocampus of Poly(I:C) mice. (a) Enzyme-linked immunosorbent assay (ELISA) measurement of the complement component 4a (C4a) in whole hippocampal homogenates from Poly(I:C) (n = 5), control (n = 5) and minocycline-treated Poly(I:C) animals (n = 4). No significant difference in C4a levels was detected between the groups. (b) Immunoreactivity for CD18 on microglia measured through the dentate gyrus (DG) of the hippocampus (DG) of Poly(I:C) (n = 7), controls (n = 5) and minocycline- treated Poly(I:C) mice (n = 4). CD18 immunoreactivity in Iba1-positive cells was significantly increased in the DG of Poly(I:C) animals as compared with controls and minocycline-treated Poly(I:C) mice. (c) Representative pictures showing the CD18 signal co-localizing with Iba1- positive cells (microglia) and showing the increased CD18 immunoreactivity in the proximity of the DG in Poly(I:C) animals as compared with controls and minocycline-treated Poly(I:C) mice. Error bars represent s.e.m. in all the panels. The data were analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls post hoc test; *Po0.05; ctrl, control animals; NS, not significant; Poly, Poly(I:C) animals; Poly/ Mino, Poly(I:C) animals treated with minocycline.

Article Snippet: Complement component 4a (C4a) levels in hippocampal lysates were measured using the mouse C4a enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology, Wuhan, China), according to the manufacturer’s manual.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Complement deposition and histopathologic changes in retinas of aged Tspan12 ECKO mice. A , Complement factor C4 deposition around retinal blood vessels, inner nuclear layer (INL), and outer plexiform layer (OPL). White arrows point to some of the small cystoid lesions decorated with complement. B , H&E staining of a control retina and a relatively strongly affected area of a mutant retina reveals cystoid lesions consistent with edema. C , Increased F4/80 staining on microglia of mutant retinas. D through F , Extravastion of IgG, transferrin, and albumin in Tspan12 ECKO retinas. G , COL4 (collagen IV) staining in retinal blood vessels of 12-mo-old ECKO mice is increased, staining in the inner limiting membrane (white arrowheads) serves as internal staining control.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Control, Mutagenesis, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Colocalization of complement factor C4 with several retinal cell types. A , Twelve-mo-old Tspan12 ECKO retinal cross-sections stained with anti-C4 and IB4. Split and merged channels are shown. Arrows indicate blood vessels decorated with C4. B , Colocalization of extravasated IgG and C4 on a subset of retinal cells and on the borders of cystoid lesions in the mutant tissue. C , Rod-bipolar cell spacing seems increased in the mutant tissue. The section shows also 1 or 2 C4-decorated Müller glia cells, which were recognized by their extensions towards the outer limiting membrane (arrow). D , Disorganized Müller cells in the mutant tissue. Sox9 marks nuclei of Müller glia. Arrow highlights a C4-decorated cell which was recognized as amacrine cell based on cell body location and cell shape. E , Partial colocalization of C4 with microglia marker IBA-1. PKC indicates protein kinase C; and Sox9, SRY-box 9.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Mutagenesis, Membrane, Marker

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A) Diagram depicting IUE surgery performed in E16 dams. (B) Representative 20X confocal image of IUE with GFP targeted to L2/3 mPFC, the electroporated region. Yellow asterisk: L2/3 GFP+ neurons. Left panel scale bar = 250 μm. Right panel scale bar = 75 μm. (C) IUE increases mC4 expression by 2.84-fold compared to P21 control. N = 18 control mice. N = 15 mC4 mice. t test with Welch’s correction. p = 0.0392. (D) mC4 mRNA expression at P60 for control and mC4 conditions. N = 10 control mice. N = 11 mC4 mice. t test with Welch’s correction. p = 0.2564. (C-D) qPCR performed from the dissected electroporated region. Control: blue. mC4: red. Mean ± SEM. (E) Immunoblot (top) and quantification (bottom) of relative mC4 levels in total lysates and isolated PSD fractions from GFP or mC4 conditions from the electroporated region. Since C4 was expressed at relatively low levels, it could only be detected when brains were pooled. Lysates and PSDs from the electroporated region were prepared from the individual mice and pooled for the western blot analysis. N = 7 mice per group for P21 (left 4 lanes). N = 4 mice per group for P60 (right 4 lanes). Vinculin was used as loading control for lysates and PSD fractions. PSD-95 immunoblot served as a control for successful isolation of PSD fractions. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . E, embryonic day; GFP, green fluorescent protein; IUE, in utero electroporation; L2/3, layer 2/3; mC4, mouse C4; mPFC, medial prefrontal cortex; P, postnatal day; PSD, postsynaptic density; qPCR, quantitative PCR.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Expressing, Control, Western Blot, Isolation, In Utero, Electroporation, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A) Developmental time course of spine density in the mPFC revealed a significant decrease in spine density (spines/μm) in neurons overexpressing C4, as compared to controls (“Con”), at P21–23. ** p < 0.01, *** p < 0.001, **** p < 0.0001. N = 240 dendrites from 84 mice (20 dendrites per each time point; 20 dendrites × 4 time points × 3 conditions). (B) Representative 40X confocal images of P21–23 apical dendritic tufts. Scale bar = 5 μm. (C) Representative 40X confocal images of P21–23 apical dendritic spine types. Yellow asterisk: large mushroom spine (TIB [a.u.] > 75%). Green arrowhead: thin spine/filopodia (TIB [a.u.] < 25%). Scale bar = 3 μm. (D) Spine density (spines/μm) sorted by spine types reveals a specific reduction of medium-sized and thin/filopodia spine types in the mC4 and hC4 condition. * p < 0.05, ** p < 0.01, **** p < 0.0001. N = 60 dendrites from 21 mice (20 dendrites per condition × 3 conditions). (E) Representative 40X confocal images of P21–23 basal dendritic spines. Scale bar = 5 μm. (F) Analysis of basal dendritic spine density (spines/μm) revealed no difference across groups. N = 80 dendrites from 28 mice (20 dendrites per each time point; 20 dendrites × 2 time points × 2 conditions). Two-way ANOVA followed by a Tukey’s test for all comparisons. Control: blue; mC4: red; hC4: purple. Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . a.u., arbitrary units; hC4, human C4; L2/3, layer 2/3; mC4, mouse C4; mPFC, medial prefrontal cortex; P, postnatal day; TIB, total integrated brightness.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Control

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A) Top: Representative whole-cell voltage-clamp recordings showing mEPSCs. Top scale bar = 250 ms/10 pA. Bottom: Same as top traces but expanded (black rectangle region). Bottom scale bar = 125 ms/10 pA. (B) Increased mC4 expression caused a reduction in mEPSC frequency. t test. **** p < 0.0001. (C) Overexpression of mC4 caused a rightward shift in the distribution of mEPSC IEI. Kolmogorov-Smirnov test. **** p < 0.0001. (D) mC4 caused a reduction in mEPSC amplitude. t test. ** p < 0.01. (E) mC4 overexpression caused a leftward shift in the mEPSC amplitude distribution. Kolmogorov-Smirnov test. ** p < 0.01. (F) mEPSC Rise 10–90 was not altered by mC4 overexpression. t test. p = 0.715. (G) No changes in mEPSC Decay tau with mC4 overexpression. t test. p = 0.07. (B-G) N = 12 control neurons, N = 10 mC4 neurons. Mean ± SEM. Control: blue; mC4: red. (H) Top: Representative recordings showing mIPSCs. Top scale bar = 250 ms/20 pA. Bottom traces are same as (H) top but expanded (rectangle region). Top scale bar = 125 ms/20 pA. (I) No difference in mIPSC frequency between control and mC4 conditions. t test. p = 0.3726. (J) Distribution of mIPSC IEIs was not changed by increased expression of mC4. Kolmogorov-Smirnov test. p > 0.05. (K) No changes in mIPSC amplitude with increased expression of mC4. t test. p = 0.5832. (L) mIPSC amplitude distribution was not changed by increased expression of mC4. Kolmogorov-Smirnov test. p > 0.05. (M) mIPSC Rise 10–90 was increased in neurons overexpressing mC4. t test. * p = 0.0329. (N) mIPSC Decay tau was increased in neurons overexpressing mC4. t test. * p = 0.0225. (I-N) N = 10 control neurons, N = 12 mC4 neurons. Control: blue; mC4: red. Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . IEI, interevent interval; mC4, mouse C4; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Expressing, Over Expression, Control

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A-B) Representative current-clamp recordings from control (A) and mC4 (B) neurons in response to injection of constant-current pulses. Voltage traces (top) shown for response to −220 pA (light blue/pink), 185 pA (medium blue/red), and 685 pA (dark blue/red) current injections (bottom). Scale bar = 500 pA or 50 mV. Scale bar = 250 ms. (C) The number of APs was not different between conditions. Dark blue trace: control mean. Light blue trace: control SEM. Dark red trace: mC4 mean. Light red trace: mC4 SEM. Two-way ANOVA. p = 0.9989. (D) Interevent interval was not different between conditions. Dark blue trace: control mean. Light blue trace: control SEM. Dark red trace: mC4 mean. Light red trace: mC4 SEM. Two-way ANOVA. p = 0.9838. (E) Rheobase was not altered by the overexpression of mC4. t test. p = 0.8795. (F) mC4 overexpression led to a dramatic reduction in C m . t test. *** p = 0.0007. (G) V m was not changed by mC4 overexpression. t test. p = 0.2166. (H) R m was not affected by mC4 overexpression. t test. p = 0.5455. (C-H) Control: N = 16 cells; mC4: N = 17 cells. Blue: control. Red: mC4. Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . AP, action potential; mC4, mouse C4.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Control, Injection, Over Expression

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A-D) Representative confocal image (60X) showing PSD-95 located within microglial lysosomes in P21 mice for control (A-B) and mC4 conditions (C-D). Single z-plane shown. Scale bar = 5 μm. Magenta: microglia (“MG) (Iba1). Red: lysosomes (CD68). Green: PSD-95 (PSD95-FingR-RFP, pseudocolored green). White arrowheads indicate colocalization of PSD95 with Iba1 (A and C) or CD68 (B and D). Panels A and C show a representative microglia (for control and mC4, respectively) including Iba1 and PSD95-FingR-RFP signal. Panels B and D show the same z-plane as panels A and C but shows CD68 and PSD95-FingR-RFP signal. Orthogonal views shown. Panels A-D each include a graph (bottom right panel) showing a line intensity scan for each signal (panels A and C show Iba1 [magenta] and PSD-95 (green); panels B and C show CD68 [red] and PSD-95 [green]). For line intensity graphs, y-axis shows gray intensity value (a.u.), and x-axis shows length (μm). (E) mC4 overexpression increased the number of microglia positive for PSD-95 engulfment (PSD95-FingR-RFP signal colocalized with CD68 and Iba1). t test. **** p < 0.0001. Red dotted line: average of control and C4 in shuffled pixel analysis. (F) There was no difference in area of PSD95 colocalized with microglia lysosomes between conditions. (MG engulfment area % = area of microglia occupied by PSD95-FingR signal in lysosomes / total microglia area). t test. p = 0.1927. (E-F) Data points represent averages from transfected region ROIs. Control: N = 26 ROIs (from 5 mice; 345 microglia). mC4: N = 26 ROIs (from 5 mice; 319 microglia). Mean ± SEM. (G-H) Microglia engulfment area (%) (from panel F) as a function of cortical depth (μm) for control (G) and mC4 (H). Data points represent individual microglia. N = 345 control and N = 319 mC4 microglia. Right graphs are same as left but are zoomed on the y-axis. Blue line: control mean. Red line: mC4 mean. Gray lines: 95% confidence intervals. Pearson’s r correlation. Control: r = 0.12. p > 0.05. mC4: r = −0.21. ** p < 0.01. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . a.u., arbitrary units; Iba1, ionized calcium binding adaptor molecule 1; mC4, mouse C4; P, postnatal day; PSD, postsynaptic density; ROI, region of interest.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Control, Over Expression, Transfection, Binding Assay

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A-D) Representative confocal images (40X) in expanded tissue showing PSD-95 within microglial lysosomes in P21 mice for control (“con”) (A and B) and mC4 (C and D) conditions. Panels A and C show a representative microglia (for control and mC4, respectively) including Iba1 and PSD95-FingR-RFP (pseudocolored green) signal. White arrowheads indicate colocalization of PSD-95 with Iba1 (A and C). Panels B and D show the same z-plane as panels A and C but with CD68 and PSD95-FingR-RFP signal. Orthogonal views shown (XY, YZ, and XZ). All images are single z-planes. Magenta: microglia (Iba1). Red: lysosomes (CD68). Green: PSD-95 (PSD95-FingR-tagRFP, pseudocolored green). Yellow box in (A and C) shows zoomed region for orthogonal views. (A and C) Left scale bar = 5 μm, right scale bar (in XY plane) = 2.5 μm. (B and D) Scale bar = 1 μm. (E) mC4 overexpression led to an increase of lysosomes that were positive for PSD-95 signal. The percent of lysosomes that were PSD95 positive per each microglia compared between control and mC4. t test. **** p < 0.0001. (F) Number of lysosomes in microglia was increased in the mC4 condition relative to controls. t test. ** p = 0.0064. (G) Lysosomes that were positive for PSD-95 were larger in size compared to PSD-95(−) lysosomes in both control (“con”) and mC4 conditions. Lysosome size (μm 2 ) per microglia for control and mC4 conditions separated into lysosomes positive or negative for PSD-95 signal. Control (+) and mC4 (+) are lysosome size for lysosomes positive for PSD95. Control (−) and mC4 (−) are lysosome size for lysosomes that are negative for PSD95. Two-way ANOVA followed by a Tukey’s test for all comparisons. Control (+ versus −): ** p = 0.0071. mC4 (+ versus −): * p = 0.0150. Control (+) versus mC4 (+): p = 0.0868. Control (−) versus mC4 (−): p = 0.2987. (E-G) Blue: control. Red: mC4. N = 86 microglia (45 control [from 5 mice] and 41 mC4 [from 4 mice] microglia). Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . ExM, expansion microscopy; Iba1, ionized calcium binding adaptor molecule 1; mC4, mouse C4; P, postnatal day; PSD, postsynaptic density.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Control, Over Expression, Microscopy, Binding Assay

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A) Representative examples of path traveled (black trace) by P18 control and mC4 pups in MI1 task. Fresh bedding corners (Fresh 1 and 2, pink) and nest bedding corner (green). (B) Control and mC4 pups spent a similar proportion of time exploring the nest and fresh corners in the MI1 task, suggesting motor and sensory skills were intact. All mice spent more time in nest bedding than in fresh bedding. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Two-way ANOVA and Sidak's posttest. Control versus mC4 time spent in nest: p > 0.9999. Control versus mC4 time spent in Fresh 1: p = 0.3327. Control versus mC4 time spent in Fresh 2: p = 0.3138. (C) Representative examples of path traveled (black trace) by P18 control and mC4 pups in MI2 task. Dam’s cup (dam: blue), empty cup (empty cup: yellow), nest bedding corner (nest: green). (D) mC4 pups spent less time interacting with the dam and more time near the empty cup compared to controls. Two-way ANOVA and Sidak's posttest. * p < 0.05. **** p < 0.0001. (E) mC4 mice took longer to approach the dam relative to controls (s). t test with Welch's correction. * p < 0.05. (F) Control and mC4 pups traveled to dam’s cup first about 50% of the time, suggesting first choice was random. Fisher’s exact test. p = 0.9999. (G) Latency to reach first cup (either dam or empty) was not different. t test with Welch's correction. p = 0.06. (H) Control and mC4 pups reached the same maximum velocity (m/s). t test. p = 0.77. (B, D-H) N = 15 control mice and N = 21 mC4 mice. Blue: control. Red: mC4. Mean ± SEM unless otherwise noted. For underlying data and tracking script, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 . mC4, mouse C4; MI1, maternal interaction 1; MI2, maternal interaction 2; P, postnatal day; Prop, proportion.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Control

Journal: PLoS Biology

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1371/journal.pbio.3000604

Figure Lengend Snippet: (A) Representative examples of path traveled (black trace) by P60 control (left) and mC4 (right) adult mice in NO interaction task. Pink corner = location of NO. (B) Control and mC4 mice spent a similar amount of time exploring the NO. Percent time spent in corner with NO shown. p = 0.2127. (C) Representative examples of path traveled (black trace) by P60 control (left) and mC4 (right) adult mice in NO recognition task. Pink corner: location of NO. Green corner: location of FO. (D) NO recognition was intact in the mC4 condition. Control and mC4 mice had a similar DI ([time with NO − time with FO] / (time with NO + time with FO]). p = 0.1539. (E) Representative examples of path traveled (black trace) by P60 control (left) and mC4 (right) adult mice in sociability task. Pink corner: location of novel mouse under mesh wire cup. Green corner: location of empty mesh wire cup. (F) mC4-overexpressing mice spent less time exploring a novel mouse and more time with the empty cup (“E”) relative to control. Graph shows DI ([time with novel mouse–time with empty cup] / [time with novel mouse + time with empty cup]). * p = 0.029. (G) Control and mC4 mice had a similar latency to first approach the novel mouse (sec). p = 0.5416. (H) Control and mC4 mice traveled to the novel mouse cup first at a similar proportion. Proportion (“Prop.”) of mice that approached the novel mouse cup first. Fisher’s exact test. p = 0.54. (I) Latency to reach first cup (either novel mouse cup or empty cup) was not different (sec) between conditions. p = 0.217. (J) Control and mC4 mice reached the same maximum velocity (m/s). p = 0.1442. N = 22 control and N = 20 mC4 mice between P60 and 70. Blue: control. Red: mC4. t test unless otherwise stated. Mean ± SEM unless otherwise noted. For underlying data and script (respectively), see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451 and https://github.com/balajisriram/dlc_utils . DI, discrimination index; FO, familiar object; mC4, mouse C4; NO, novel object; P, postnatal day.

Article Snippet: DNA sequences containing m C4b (NM_009780.2, synthesized by Genescript) and h C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: Control