Journal: Brain, behavior, and immunity

Article Title: HIV-1 Tat protein enhances sensitization to methamphetamine by affecting dopaminergic function

doi: 10.1016/j.bbi.2017.05.004

Figure Lengend Snippet: Immunohistochemistry on paraffin embedded sections was utilized examine the protein distribution and levels of dopamine receptor D1 (A, B, C, D), dopamine receptor D2 (E, F, G, H), as well as of IBA-1 (I, J, K, L) in SAL TAT− (A, E, I), SAL TAT+ (B, F, J), METH TAT− (C, G, K), and METH TAT+ (D, H, L) mice. Representative positive cells in the 40× magnification images were labeled with a black arrow. (M) Normalized intensity density was calculated in ImageJ. Data are expressed as Mean ± SEM (n=5). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were blocked with 5g/l Casein (Sigma Aldrich) in PBS, containing 0.5g/l Thimerosal (Sigma Aldrich) and incubated with Iba-1 antibody (Wako Lab Chemicals, Richmond, VA), the anti-mouse DRD1 antibody (NLS43, Novus Biologicals, Littleton, CO), or anti mouse DRD2 (orb154598, Biorbyt, San Francisco, CA), each one diluted in Casein buffer.

Techniques: Immunohistochemistry, Labeling

Journal: Theranostics

Article Title: Dopamine receptor 1 on CaMKII-positive neurons within claustrum mediates adolescent cocaine exposure-induced anxiety-like behaviors and electro-acupuncture therapy

doi: 10.7150/thno.83079

Figure Lengend Snippet: ACE enhances anxiety-like behaviors in mice during adulthood, along with the increased CaMKII-positive neuron activity and D1R CaMKII level in claustrum. A , Experimental timeline. B , EPM test. n = 16 mice per group. C , Total protein levels of c-Fos. n = 3 mice per group. D , The number and percentage of c-Fos-positive & CaMKII-positive neurons. n = 6 mice per group. Scale bar, 100 μm/50 μm. E , The mRNA levels of d1r, d2r and d3r . n = 3 mice per group. F , The protein levels of D1R, D2R and D3R. n = 3 mice per group. G , The correlation analysis of claustrum D1R levels with EPM data. n = 6 mice. H , The expression of D1R CaMKII . n = 6 mice per group. Scale bar, 100 μm/50 μm. I , Levels of p-ERK 1/2 and ERK 1/2. n = 3 mice per group. J , Levels of p-CREB, CREB and BDNF. n = 3 mice per group. ASE, adolescent saline exposure; ACE, adolescent cocaine exposure; CL, claustrum. Data are presented as the Mean ± SEM. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 vs ASE.

Article Snippet: The transferred membranes were blocked with 5% non-fat dry milk or 5% BCA and 0.1% Tween 20 in 10 mM Tris-HCl (TBST buffer) for 1.5 h at room temperature, then subsequently incubated with the following primary antibodies: D1R (1:1000, Rabbit, RRID:AB_10598308, Proteintech, USA), D2R (1:1000, Rabbit, ER1907-70, Hua An Biotechnology Company, China), D3R (1:1000, Mouse, ab155098, Abcam, UK), ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), p-ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), CREB (1:1000, Rabbit, RRID:AB_10699020, Cell Signaling Technology, USA), p-CREB (1:1000, Rabbit, RRID:AB_2561044, Cell Signaling Technology, USA), c-Fos (1:2000, Rabbit, RRID:AB_2247211, Cell Signaling Technology, USA), BDNF (1:1000, Mouse, RRID:AB_2881675, Proteintech, USA).

Techniques: Activity Assay, Expressing, Saline

Journal: Theranostics

Article Title: Dopamine receptor 1 on CaMKII-positive neurons within claustrum mediates adolescent cocaine exposure-induced anxiety-like behaviors and electro-acupuncture therapy

doi: 10.7150/thno.83079

Figure Lengend Snippet: Inhibiting claustrum D1R suppresses ACE-induced anxiety-like behaviors in mice during adulthood and claustrum activation. A , Experimental timeline for SCH-23390 treatment and diagram of cannula implantation in claustrum. Scale bar, 400 μm. B , EPM test. Vehicle-treated ASE mice, n = 6 mice; SCH-23390-treated ASE mice, n = 6 mice; Vehicle-treated ACE mice, n = 10 mice; SCH-23390-treated ACE mice, n = 8 mice. C , The number of c-Fos-positive neurons in claustrum. n = 6 mice per group. Scale bar, 100 μm. D , Experimental timeline for whole-cell patch in claustrum slices. E , Action potentials (AP) of claustrum neurons under whole-cell current-clamp configuration (n = 16 cells from 4 mice) and sample traces for the number of AP following 200 pA current injection in the absence and presence of SCH-23390 on the same cell. Veh, vehicle-treated mice; SCH, SCH-23390-treated mice; CL, claustrum. Data are presented as the Mean ± SEM. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 vs Veh or Baseline.

Article Snippet: The transferred membranes were blocked with 5% non-fat dry milk or 5% BCA and 0.1% Tween 20 in 10 mM Tris-HCl (TBST buffer) for 1.5 h at room temperature, then subsequently incubated with the following primary antibodies: D1R (1:1000, Rabbit, RRID:AB_10598308, Proteintech, USA), D2R (1:1000, Rabbit, ER1907-70, Hua An Biotechnology Company, China), D3R (1:1000, Mouse, ab155098, Abcam, UK), ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), p-ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), CREB (1:1000, Rabbit, RRID:AB_10699020, Cell Signaling Technology, USA), p-CREB (1:1000, Rabbit, RRID:AB_2561044, Cell Signaling Technology, USA), c-Fos (1:2000, Rabbit, RRID:AB_2247211, Cell Signaling Technology, USA), BDNF (1:1000, Mouse, RRID:AB_2881675, Proteintech, USA).

Techniques: Activation Assay, Injection

Journal: Theranostics

Article Title: Dopamine receptor 1 on CaMKII-positive neurons within claustrum mediates adolescent cocaine exposure-induced anxiety-like behaviors and electro-acupuncture therapy

doi: 10.7150/thno.83079

Figure Lengend Snippet: Knocking-down claustral D1R CaMKII level suppresses ACE-induced anxiety-like behaviors in mice during adulthood and claustrum activation. A , Experimental design and timeline for KD virus treatment. B , Local injections of virus into claustrum. Scale bar, 300 μm. C , The D1R protein level. n = 3 mice per group. D , EPM test. Control virus-treated ASE mice, n = 9 mice; Knocking-down virus-treated ASE mice, n = 9 mice; Control virus-treated ACE mice, n = 7 mice; Knocking-down-treated ACE mice, n = 9 mice. E , The percentage of c-Fos-positive neurons on EGFP-transfected neurons. Control virus-treated ASE mice, n = 6 mice; Knocking-down virus-treated ASE mice, n = 6 mice; Control virus-treated ACE mice, n = 4 mice; Knocking-down-treated ACE mice, n = 6 mice. Scale bar, 100 μm/50 μm. Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice; CL, claustrum. Data are presented as the Mean ± SEM. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 vs Ctrl.

Article Snippet: The transferred membranes were blocked with 5% non-fat dry milk or 5% BCA and 0.1% Tween 20 in 10 mM Tris-HCl (TBST buffer) for 1.5 h at room temperature, then subsequently incubated with the following primary antibodies: D1R (1:1000, Rabbit, RRID:AB_10598308, Proteintech, USA), D2R (1:1000, Rabbit, ER1907-70, Hua An Biotechnology Company, China), D3R (1:1000, Mouse, ab155098, Abcam, UK), ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), p-ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), CREB (1:1000, Rabbit, RRID:AB_10699020, Cell Signaling Technology, USA), p-CREB (1:1000, Rabbit, RRID:AB_2561044, Cell Signaling Technology, USA), c-Fos (1:2000, Rabbit, RRID:AB_2247211, Cell Signaling Technology, USA), BDNF (1:1000, Mouse, RRID:AB_2881675, Proteintech, USA).

Techniques: Activation Assay, Virus, Control, Transfection

Journal: Theranostics

Article Title: Dopamine receptor 1 on CaMKII-positive neurons within claustrum mediates adolescent cocaine exposure-induced anxiety-like behaviors and electro-acupuncture therapy

doi: 10.7150/thno.83079

Figure Lengend Snippet: EA treatment rescues ACE-increased anxiety-like behaviors, claustrum activation and D1R CaMKII level during adulthood. A , Experimental design and timeline for EA treatment. B , Schematic diagram of EA and SEA treatments in ACE mice. C , EPM test. EA group, n = 10 mice; SEA group, n = 13 mice. D , The number and percentage of c-Fos-positive & CaMKII-positive neurons. n = 6 mice per group. Scale bar, 100 μm/50 μm. E , The D1R protein level. n = 3 mice per group. F , The expression of D1R CaMKII . n = 6 mice per group. Scale bar, 100 μm/50 μm. G , The correlation analysis of claustrum D1R level with EPM data. n = 6 mice. H , Levels of p-ERK 1/2 and ERK 1/2 . I , Levels of p-CREB, CREB and BDNF. n = 3 mice per group. SEA, sham electro-acupuncture-treated ACE mice; EA, electro-acupuncture (GV20 and GV29)-treated ACE mice; CL, claustrum. Data are presented as the Mean ± SEM. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 vs SEA.

Article Snippet: The transferred membranes were blocked with 5% non-fat dry milk or 5% BCA and 0.1% Tween 20 in 10 mM Tris-HCl (TBST buffer) for 1.5 h at room temperature, then subsequently incubated with the following primary antibodies: D1R (1:1000, Rabbit, RRID:AB_10598308, Proteintech, USA), D2R (1:1000, Rabbit, ER1907-70, Hua An Biotechnology Company, China), D3R (1:1000, Mouse, ab155098, Abcam, UK), ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), p-ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), CREB (1:1000, Rabbit, RRID:AB_10699020, Cell Signaling Technology, USA), p-CREB (1:1000, Rabbit, RRID:AB_2561044, Cell Signaling Technology, USA), c-Fos (1:2000, Rabbit, RRID:AB_2247211, Cell Signaling Technology, USA), BDNF (1:1000, Mouse, RRID:AB_2881675, Proteintech, USA).

Techniques: Activation Assay, Expressing

Journal: Theranostics

Article Title: Dopamine receptor 1 on CaMKII-positive neurons within claustrum mediates adolescent cocaine exposure-induced anxiety-like behaviors and electro-acupuncture therapy

doi: 10.7150/thno.83079

Figure Lengend Snippet: Schematic summary of present study. Adolescent cocaine exposure (ACE) enhances anxiety-like behaviors in male mice during adulthood, accompanied with triggered activation of CaMKII-positive neurons and more dopamine receptor 1 (D1R) on CaMKII-positive neurons (D1R CaMKII ) in claustrum. Either blocking D1R with SCH-23390 or knocking-down D1R CaMKII with virus in the claustrum efficiently reduces claustrum activation, and ultimately suppresses ACE-induced anxiety-like behaviors in mice. By targeting claustrum D1R CaMKII , electro-acupuncture (EA) at acupoints of GV20 and GV29 rescues ACE-increased anxiety-like behaviors in mice during adulthood. In the process, the p-CREB and BDNF may be the potential subsequent signals to D1R CaMKII .

Article Snippet: The transferred membranes were blocked with 5% non-fat dry milk or 5% BCA and 0.1% Tween 20 in 10 mM Tris-HCl (TBST buffer) for 1.5 h at room temperature, then subsequently incubated with the following primary antibodies: D1R (1:1000, Rabbit, RRID:AB_10598308, Proteintech, USA), D2R (1:1000, Rabbit, ER1907-70, Hua An Biotechnology Company, China), D3R (1:1000, Mouse, ab155098, Abcam, UK), ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), p-ERK 1/2 (1:1000, Rabbit, BM4326, Boster, China), CREB (1:1000, Rabbit, RRID:AB_10699020, Cell Signaling Technology, USA), p-CREB (1:1000, Rabbit, RRID:AB_2561044, Cell Signaling Technology, USA), c-Fos (1:2000, Rabbit, RRID:AB_2247211, Cell Signaling Technology, USA), BDNF (1:1000, Mouse, RRID:AB_2881675, Proteintech, USA).

Techniques: Activation Assay, Blocking Assay, Virus

Journal: Translational Cancer Research

Article Title: DRD1 and DRD4 are differentially expressed in breast tumors and breast cancer stem cells: pharmacological implications

doi: 10.21037/tcr-22-783

Figure Lengend Snippet: Expression of DRs breast cancer cell lines. (A) mRNA expression of DRDs in 62 breast cancer cell lines form the Cancer Cell Line Encyclopedia. MCF-7 and MDA-MB-231 cell lines are shown in magenta and red, respectively. (B,C) Protein quantification of DRD1, DRD2, and DRD4 in MCF-7 (B) and MDA-MB-231 (C) by flow cytometry. Graphs in (B) and (C) show MFI (average ± SEM) from 2–3 independent experiments. **, P<0.05 (Student’s t- test). DR, dopamine receptor; MFI, mean fluorescence intensity; SEM, standard error of the mean; TPM, transcript per million.

Article Snippet: Expression of DRs was analyzed using anti-human DRD1 Alexa Fluor 405 (FAB8276V, R&D Systems), anti-human DRD2 Alexa Fluor 647 (sc-5303, Santa Cruz Biotechnology), or anti-human DRD4 PE (sc-136169, Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Psychopharmacology

Article Title: Extracellular Dopamine, Acetylcholine, and Activation of Dopamine D1 and D2 Receptors after Selective Breeding for Cocaine Self-Administration in Rats

doi: 10.1007/s00213-017-4640-7

Figure Lengend Snippet: Counts of cells expressing c-Fos, D1R and D2R in different brain regions of LS and HS rats. Values indicate the number of cells per field, scored under blinded conditions, with data shown for five animals per condition.

Article Snippet: Sections were then incubated with sheep anti-c-Fos (1:500, CBL440, Millipore, Temecula, USA), combined with either mouse anti-Dopamine D1R (1:1000, NB110, Novus Biologicals, Littleton, USA) or rabbit anti-Dopamine D2R (1:500, AB5084P, Millipore, Temecula, USA) antibodies over 48 h in a humidi ed chamber at 4°.

Techniques: Expressing

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: D1r downregulation in adolescence is associated with complement C3 and microglial engulfment in males, but not the females. Triple-label immunohistochemistry for D1r, C3, and Iba1 was performed and protein expression levels for each target were normalized for z-stack size. Because male and female tissue was processed at different times and required different image acquisition parameters, neither data nor images can be directly compared between the sexes. Representative images precede histograms; scale bars are equal to 20 µm. Males: a D1r levels transiently increased at P30 (Table ; b C3 levels decreased after P30 (Table ; c Iba1 levels decreased after P30 (Table . d Merged male images. Females: e D1r levels decreased after P20 (Table f C3 levels transiently increased at P38 (Table ; g Iba1 levels decreased after P20 (Table . h Merged female images. Images were then analyzed using a volumetric reconstruction of Iba1 (i.e., microglia) as a mask to determine the volume of D1r, C3, or colocalized C3-D1r physically associated with (either in contact with or inside) microglia. Masked volumes were normalized for total protein of the target and total Iba1 (see Methods). i Representative 2D triple-label immunohistochemistry (scale bar 20 µm), with enlarged 3D representation (scale bar 3 µm) of the selected area demonstrating (left-right) D1r, C3, and C3-D1r contacted by/internal to the microglia. Open arrow head indicates a region of the microglia where both D1r and C3 are located, but not colocalized, demonstrating the sensitivity of colocalization analyses. Closed arrow head indicates a region of the microglia where D1r and C3 colocalize (C3-D1r). Males: j D1rs are maximally contacted by microglia (irrespective of changing D1r and Iba1 levels) at P30 (Table ). k C3 contact by microglia does not change over time (Table ), and L C3-D1r contact is high between P30-P38 (Table ). Females: m D1rs are maximally contacted by microglia at P54, not at P20, prior to their elimination (Table ). n C3 contact is also increased at P54 (Table ), while o C3-D1r contact does not change over development (Table )

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: D1r downregulation in adolescence is associated with complement C3 and microglial lysosomal degradation in the male, but not the female, NAc. To assess microglial phagocytic activity and whether D1r, C3, or C3-D1r were localized in microglial CD68+ lysosomes, immunohistochemistry for CD68, D1r, and C3 was performed. a Representative 2D triple-label immunohistochemistry (scale bar 20 µm), with enlarged 3D representation (scale bar 2 µm) of the selected area demonstrating (left-right) D1r, C3, and C3-D1r within CD68+ lysosomes. Closed arrow head indicates a lysosome where D1r and C3 colocalize (C3-D1r). Males: c CD68 levels are high from P30 to 38 (Table ). b D1r content in CD68+ lysosomes is highest at P30 (Table ), d C3 content in lysosomes does not change over development (Table ), and e C3-D1r content in lysosomes is transiently elevated at P30 (Table ). Females: f CD68 levels are transiently elevated at P30 (Table ). g D1r content in CD68+ lysosomes is high at both P30 and P54 (Table ), h C3 content in lysosomes is highest at P20 and P54 (Table ), and i C3-D1r content in lysosomes does not change over development (Table ). For all experiments, n = 4 animals/sex/group. Data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Activity Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: C3–C3R interactions mediate developmentally-typical D1r elimination in vivo in males, but not females. Neutrophil inhibitor factor (NIF), a peptide that binds specifically to the CD11b subunit of C3 receptors (C3Rs), was assessed for its efficacy in reducing microglial phagocytic activity ex vivo. a In microglia isolated from whole brain, 60 ng, but not 120 ng NIF inhibited microglial phagocytosis of fluorescent beads (Table ). Representative images precede histograms; scale bar equals 20 µm. n = 4, with 2 replications/condition. b Microglia were isolated from whole brain and incubated with 60 ng NIF, and then immunocytochemically assessed for NIF and CD11b at 30, 60, and 120 min. Closed arrow heads indicate NIF (green) immunoreactivity. Scale bar equals 10 µm. There was no change in c NIF or (Table ) d CD11b (Table ) immunoreactivity over 2 h, suggesting that NIF was not causing the degradation of its receptor. n = 3; 2 replications/condition. To determine if developmental D1r downregulation requires C3–C3R interactions, NIF or Vehicle was microinjected in the NAc at e P30 in males and g P22 in females (both represent sex-specific ages prior to D1r downregulation), and then tissue was assessed at P38 and P30, respectively, an age at which D1rs should be developmentally downregulated. f In males, NIF-treated hemispheres exhibited significantly more (~25%) D1r immunoreactivity than within-animal vehicle-treated hemispheres (Table ). h In females, NIF-treated hemispheres exhibited the same D1r immunoreactivity as within-animal vehicle-treated hemispheres (Table ). Representative images precede histograms; scale bars equal 100 µm. n = 4/sex with counterbalanced injections. For ex vivo experiments, data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. For in vivo experiments, D1r data from 4–7 different sections per animal were averaged and then calculated as a percentage of within-animal vehicle control levels. Data were analyzed with 2-tailed one-sample t -tests. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( a – d ) and one-sample t -tests from 100 ( f , h ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: In Vivo, Activity Assay, Ex Vivo, Isolation, Incubation, Control

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Sex-specific social behavior patterns over adolescence require C3–C3R interactions. a Experimental design: separate cohorts of experimental animals ( n = 8/sex/age) were single-housed for 24 h prior to test, after which time a novel age-matched and sex-matched conspecific was introduced into their home cage. Ten minutes of interactions were recorded, and later an experimenter blinded to the conditions coded the interactions for either total play or social exploration (i.e., non-play social behavior). Only behaviors initiated by the experimental animal were considered. Males: b Male social play behavior transiently increased at P30 (Table ), c with no changes in social exploration over development (Table ). Females: d Female social play behavior did not change over development (Table ), e while social exploration peaked from P30 to 38 (Table ). f To determine if C3–C3R interactions regulating D1r levels between P30 and 38 in males is required for the decline in social play behavior observed at this time, NIF or vehicle was injected bilaterally in P30 males or P22 females ( n = 10/sex/treatment). Animals were then single housed for 24 h at P37 or P29, and assessed for social behavior as described at P38 and P30, respectively. Males: g Interrupting C3–C3R interactions at P30 increased social play behavior at P38 (Table ), h without affecting social exploration (Table ). Females: i Female social play behavior, while at a low basal level, also modestly increased at P30 after P22 NIF injection (Table , j with no changes in social exploration (Table ). Data were analyzed either with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons or with two-tailed unpaired t -tests. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( b – e ) and unpaired t -tests ( g – j ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: C3–C3R regulation of D1rs regulates male, but not female, social play behavior. To determine if disrupting C3–C3R interactions via NIF increases play in a D1r-dependent manner, we utilized siRNA against rat D1r (200 µM) or scRNA controls. a P30 males were co-injected with NIF + D1r siRNA in one hemisphere and NIF + scRNA in the contralateral hemisphere, and then D1r immunoreactivity was assessed at P38. b In the presence of NIF, D1r siRNA was capable of decreasing D1r levels relative to NIF + scRNA within-animal control hemispheres (~20%; Table ). c P22 females were injected with NIF + D1r siRNA in one hemisphere and NIF + scRNA in the contralateral hemisphere, and then D1r immunoreactivity was assessed at P30. d In the presence of NIF, D1r siRNA was capable of decreasing D1r levels relative to NIF + scRNA within-animal control hemispheres (~30%; Table ). Representative images precede histograms; scale bar equals 100 µm. n = 3/sex. e Experimental design: P30 males and P22 females were bilaterally microinjected into the NAc with either Vehicle + scRNA (Veh sc), Vehicle + D1r siRNA (Veh si), NIF + scRNA (NIF sc), or NIF + D1r siRNA (NIF si). At P37 and P22, animals were single-housed for 24 h, and then a novel age- and sex-matched conspecific was introduced into their home cage for 10 min social behavior tests ( n = 9–10/sex/treatment). Males: f NIF + scRNA increased social play behavior in males, which was eliminated by D1r siRNA (NIF si; Table ). g No treatment changed social exploration behavior (Table ). Females: h NIF + scRNA increased social play behavior in females, which was not affected by D1r siRNA (NIF si; Table ). i No treatment changed social exploration behavior (Table ). Immunohistochemical data were calculated as in Fig. and analyzed with two-tailed one-sample t -tests. Behavioral data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. Histograms portray the mean ± SEM with individual data points overlaid. Significant one-sample t -tests from 100 ( b , d ) and post-hoc Holm-Sidak t -tests ( f – i ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Injection, Control, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 1

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Comparison

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 2

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Comparison

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 5

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Comparison, In Vivo, Injection

Journal: ASN neuro

Article Title: Forelimb Motor Skills Deficits Following Thoracic Spinal Cord Injury: Underlying Dopaminergic and Neural Oscillatory Changes in Rat Primary Motor Cortex.

doi: 10.1177/17590914211044000

Figure Lengend Snippet: Figure 6. Thoracic SCI reduces DA levels, D1R and D2R expression in M1. DA levels and expression of dopamine receptors (D1R and D2R) were determined in contralateral FLA (Cont-FLA), ipsilateral FLA (Ipsi-FLA), and contralateral HLA (Cont-HLA) of the untrained Control (UT-Control) subgroup, and both trained Control and SCI main groups. Bar diagram demonstrates DA levels of cortical areas in all groups (A). A representative immunoblotting bands of D1R and D2R (B). Bar diagrams show D1R (C) and D2R (D) levels (normalized against β actin) of cortical areas in all groups. Data are presented as means ± SEM (n = 5 per group). Denotation of significant differences between all three groups (*P <.05, **P <.01, ***P <.001). Denotation of significant differences within-group comparison with respect to Cont-FLA ($P <.05, $$P <.01, $$$P <.001) and Ipsi-FLA (#P <.05, ##P <.01). Note. SCI = spinal cord injury; DA = dopamine; HLA = hindlimb area; FLA = forelimb area.

Article Snippet: The membrane was gently shaken with blocking buffer containing 2% non-fat dry milk in TBS-T (50 mM Tris–HCl, 0.9% NaCl, 0.1%Tween-20, PH 7.5) for 1 h at room temperature and then incubated with monoclonal primary antibodies raised against D1R (mouse, 1:200; Cat.No: sc-33660, RRID: AB_668813, Santa Cruz Biotechnology, USA) and D2R (mouse, 1:200; Cat.No: sc-5303, RRID: AB_668816, Santa Cruz Biotechnology, USA). β-actin antibody (mouse, 1:200; Cat.No: sc-69879, RRID: AB_1119529, Santa Cruz Biotechnology, USA) was performed as a loading control.

Techniques: Expressing, Control, Western Blot, Comparison

Journal: ASN neuro

Article Title: Forelimb Motor Skills Deficits Following Thoracic Spinal Cord Injury: Underlying Dopaminergic and Neural Oscillatory Changes in Rat Primary Motor Cortex.

doi: 10.1177/17590914211044000

Figure Lengend Snippet: Figure 7. Multiple correlations among motor skills, DAergic markers and oscillatory powers. Day-by-day correlations between Cont-FLA oscillatory powers during task condition and the first reach success rate for each rat (A). Animal-by-animal correlations between resting state oscillatory powers (day 6) and DA/D1R/D2R levels in the Cont-FLA and Cont-HLA (B). Note: Bordered squares indicate significant correlation. The color bar is ranged from blue (strong negative correlation) to red (strong positive correlation). Animal-by-animal correlations between learning rate for first reach success and DA/ D1R/ D2R levels in Cont-FLA (C) and Ipsi-FLA (D). Note. DA = dopamine; FLA = forelimb area; HLA = hindlimb area.

Article Snippet: The membrane was gently shaken with blocking buffer containing 2% non-fat dry milk in TBS-T (50 mM Tris–HCl, 0.9% NaCl, 0.1%Tween-20, PH 7.5) for 1 h at room temperature and then incubated with monoclonal primary antibodies raised against D1R (mouse, 1:200; Cat.No: sc-33660, RRID: AB_668813, Santa Cruz Biotechnology, USA) and D2R (mouse, 1:200; Cat.No: sc-5303, RRID: AB_668816, Santa Cruz Biotechnology, USA). β-actin antibody (mouse, 1:200; Cat.No: sc-69879, RRID: AB_1119529, Santa Cruz Biotechnology, USA) was performed as a loading control.

Techniques: