Journal: bioRxiv

Article Title: Early life stress impairs VTA coordination of BLA network and behavioral states

doi: 10.1101/2023.09.16.558081

Figure Lengend Snippet: ( A ) Representative 20X image for CNT (Left) and ELS (Right) BLA slice, outlined in white. DAPI: blue, D1 receptor (D1R) immunoreactivity: green. ( B ) Summary plot shows mean BLA nuclei count in CNT (N = 5 animals, n = 6 slice per animal) and ELS (N = 4 animals, n = 6 slice per animal). ( C ) Summary plot shows mean optical density of BLA D1Rs for CNT and ELS animals. ( D ) Left: Representative images of coronal hindbrain slices for a CNT (Top) and ELS (Bottom) animal at 4X. Right: 20x images of outlined rectangles on 4X images with arrow-tips indicating tdT-TH+ colocalized neurons; VTA: white square; substantia nigra (SN): white rectangle. ( E ) Top: Grouped bar plot shows average number of BLA-projecting neurons (tdT+) from SN and VTA. Bottom: Grouped bar plot shows average number of TH+ neurons in SN and VTA. ( F ) Grouped bar plot shows percentage of tdT-TH+ colocalized neurons in SN and VTA. ** p < 0.01. Error bars represent SEM.

Article Snippet: D1-receptor expression : Primary; chicken anti-DA receptor D1a (1:600; Millipore MAB5290) TH expression: Primary; chicken anti-TH primary antibody (1:1000; Abcam, AB76442) A wide-field epifluorescence microscope (Keyence BZ-X700) was used to acquire images of mounted sections.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Dopamine D2 receptor on CD4 + T cells is protective against inflammatory responses and signs in a mouse model of rheumatoid arthritis

doi: 10.1186/s13075-023-03071-1

Figure Lengend Snippet: Effects of global D1r or D2r ablation on differentiation and function of CD4 + T cells in CIA mice. A Protein expression of D1R and D2R in ankle joints and spleen of mice. The left panel indicates Western blotting bands and the right panel is densitometric data normalized to β-actin ( n = 5/group). B Flow cytometric assay of the CD4 + T cell subsets, CD4 + IFN-γ + (Th1) cells, CD4 + IL-4 + (Th2) cells, CD4 + IL-17 + (Th17) cells, and CD4 + CD25 + Foxp3 + (Treg) cells. Representative images of the flow cytometric assay are exhibited in the left panel and quantitative data showing percentages of the CD4 + T cell subsets in total CD4 + T cells are indicated in the right panel ( n = 5/group). C Protein expression of the specific transcription factors T-bet, GATA-3, ROR-γt and Foxp3 for Th1, Th2, Th17 and Treg cells, respectively, in ankle joints. Representative Western blotting bands are exhibited in the left panel and densitometric data normalized to β-actin are indicated in the right panel ( n = 5/group). D-G Contents of the cytokines IFN-γ, IL-4, IL-17A and TGF-β1 in ankle joints measured by ELISA. n = 6/group. ** P < 0.01, versus control or WT mice; # P < 0.05, ## P < 0.01, versus WT CIA mice

Article Snippet: The membranes were blocked with 5% skim milk and separately incubated with the primary rabbit antibodies specific for D1R (1:200; Millipore, USA), D2R (1:200; Millipore, USA), T-bet (1:200; Santa Cruz Biotechnology, USA), GATA-3 (1:200; Santa Cruz Biotechnology, USA), ROR-γt (1:200; Invitrogen, USA), or Foxp3 (1:200; Santa Cruz Biotechnology, USA), or with the primary mouse antibody specific for β-actin (1:5000; Sigma-Aldrich, USA) overnight at 4 °C.

Techniques: Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Molecular Pain

Article Title: Dopamine receptor D2, but not D1, mediates the reward circuit from the ventral tegmental area to the central amygdala, which is involved in pain relief

doi: 10.1177/17448069221145096

Figure Lengend Snippet: Pain relief increased dopaminergic D2 receptor expression in injured mice. D2 receptor expression in the CeA was significantly decreased in incised mice treated with PNB compared with incised mice treated with saline (n = 6; * p < 0.05 vs the incision+saline group; one-way ANOVA,Interaction::F (3, 20) = 6.274). The expression of D1 receptor was not significantly changed. (b) In the CeA, D1 receptor was mainly expressed in glutamatergic excitatory neurons (positive for glutaminase). In the CeA, D2 receptor was predominantly expressed in GABAergic inhibitory neurons (positive for GABA). Scale bar, 50 μm.

Article Snippet: Anti-D1 receptor , 1:200 , Millipore , MAB5290.

Techniques: Expressing

Journal: Molecular Pain

Article Title: Dopamine receptor D2, but not D1, mediates the reward circuit from the ventral tegmental area to the central amygdala, which is involved in pain relief

doi: 10.1177/17448069221145096

Figure Lengend Snippet:

Article Snippet: Anti-D1 receptor , 1:200 , Millipore , MAB5290.

Techniques:

Journal: Molecular Pain

Article Title: Dopamine receptor D2, but not D1, mediates the reward circuit from the ventral tegmental area to the central amygdala, which is involved in pain relief

doi: 10.1177/17448069221145096

Figure Lengend Snippet: Pain relief increased dopaminergic D2 receptor expression in injured mice. D2 receptor expression in the CeA was significantly decreased in incised mice treated with PNB compared with incised mice treated with saline (n = 6; * p < 0.05 vs the incision+saline group; one-way ANOVA,Interaction::F (3, 20) = 6.274). The expression of D1 receptor was not significantly changed. (b) In the CeA, D1 receptor was mainly expressed in glutamatergic excitatory neurons (positive for glutaminase). In the CeA, D2 receptor was predominantly expressed in GABAergic inhibitory neurons (positive for GABA). Scale bar, 50 μm.

Article Snippet: After being blocked with 3% bovine serum albumin (BSA) (Solarbio, Beijing, China) and incubated overnight at 4°C with primary antibodies against D1R (1:1000; catalog No. MAB5290, Millipore), D2R (1:1000 catalog No. AB5084P, Millipore), and GAPDH (catalog No. RM2002 L, Beijing Ray), the membranes were washed six times for 10 min each in Tris-buffered saline and 1% Tween.

Techniques: Expressing

Journal: Molecular Pain

Article Title: Dopamine receptor D2, but not D1, mediates the reward circuit from the ventral tegmental area to the central amygdala, which is involved in pain relief

doi: 10.1177/17448069221145096

Figure Lengend Snippet:

Article Snippet: After being blocked with 3% bovine serum albumin (BSA) (Solarbio, Beijing, China) and incubated overnight at 4°C with primary antibodies against D1R (1:1000; catalog No. MAB5290, Millipore), D2R (1:1000 catalog No. AB5084P, Millipore), and GAPDH (catalog No. RM2002 L, Beijing Ray), the membranes were washed six times for 10 min each in Tris-buffered saline and 1% Tween.

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: Targeting the D1-N-methyl- d -aspartate receptor complex reduces l -dopa-induced dyskinesia in 6-hydroxydopamine-lesioned Parkinson’s rats

doi: 10.2147/DDDT.S93487

Figure Lengend Snippet: Effects of intrastriatal administration of Tat-fusion interfering peptide (Tat-D1Ri) on D1R–GluN1 interactions. Notes: Tat-D1Ri, Tat-D1Rc, or saline were locally injected into the striatum of the normal adult rat at a rate of 0.2 µL/min. Rat striatal tissues were then used for coimmunoprecipitation experiments to validate the efficacy and selectivity of interfering peptide. The intrastriatal injection of Tat-D1Ri rather than Tat-D1Rc caused reduction of D1R–GluN1 interactions, which demonstrated the effectiveness of Tat-D1Ri. Abbreviations: Tat-D1Rc, Tat-fusion control peptide; Ig, Immunoglobulin; IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Samples were incubated with a rabbit antibody against D1R (EMD Millipore, Billerica, MA, USA) or a mouse antibody against GluN1 (Millipore) overnight at 4°C.

Techniques: Injection, Immunoprecipitation, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Targeting the D1-N-methyl- d -aspartate receptor complex reduces l -dopa-induced dyskinesia in 6-hydroxydopamine-lesioned Parkinson’s rats

doi: 10.2147/DDDT.S93487

Figure Lengend Snippet: Effects of intrastriatal administration of Tat-D1Ri on membrane D1R subunit expression. Notes: Protein levels were evaluated by Western blotting of proteins extracted from the lesioned striatum of the rat brains. They were assessed in extracts from 6-OHDA-lesioned rats treated with pulsatile l -dopa plus intrastriatal administration of Tat-D1Rc or Tat-D1Ri. ( A ) Representative Western blot analysis of D1R in the membrane fraction; ( B ) densitometric analysis of two blots with specific protein signals normalized to the corresponding GAPDH levels. Data are expressed as means ± SEM. Statistical analysis was conducted by independent-samples t -test. * P =0.05 versus LID + Tat-D1Rc. Abbreviations: 6-OHDA, 6-hydroxydopamine; l -dopa, L-3,4-dihydroxyphenylalanine; LID, l -dopa-induced dyskinesia; SEM, standard error of mean; Tat-D1Ri, Tat-fusion interfering peptide; Tat-D1Rc, Tat-fusion control peptide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Samples were incubated with a rabbit antibody against D1R (EMD Millipore, Billerica, MA, USA) or a mouse antibody against GluN1 (Millipore) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Targeting the D1-N-methyl- d -aspartate receptor complex reduces l -dopa-induced dyskinesia in 6-hydroxydopamine-lesioned Parkinson’s rats

doi: 10.2147/DDDT.S93487

Figure Lengend Snippet: Effects of intrastriatal administration of Tat-fusion interfering peptide (Tat-D1Ri) on D1R–GluN1 interactions. Notes: Tat-D1Ri, Tat-D1Rc, or saline were locally injected into the striatum of the normal adult rat at a rate of 0.2 µL/min. Rat striatal tissues were then used for coimmunoprecipitation experiments to validate the efficacy and selectivity of interfering peptide. The intrastriatal injection of Tat-D1Ri rather than Tat-D1Rc caused reduction of D1R–GluN1 interactions, which demonstrated the effectiveness of Tat-D1Ri. Abbreviations: Tat-D1Rc, Tat-fusion control peptide; Ig, Immunoglobulin; IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Membranes were blocked in 5% nonfat milk for 1 hour at room temperature and incubated with a rabbit primary antibody against D1R (Millipore) or a mouse primary antibody against GluN1 (Millipore) overnight at 4°C.

Techniques: Injection, Immunoprecipitation, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Targeting the D1-N-methyl- d -aspartate receptor complex reduces l -dopa-induced dyskinesia in 6-hydroxydopamine-lesioned Parkinson’s rats

doi: 10.2147/DDDT.S93487

Figure Lengend Snippet: Effects of intrastriatal administration of Tat-D1Ri on membrane D1R subunit expression. Notes: Protein levels were evaluated by Western blotting of proteins extracted from the lesioned striatum of the rat brains. They were assessed in extracts from 6-OHDA-lesioned rats treated with pulsatile l -dopa plus intrastriatal administration of Tat-D1Rc or Tat-D1Ri. ( A ) Representative Western blot analysis of D1R in the membrane fraction; ( B ) densitometric analysis of two blots with specific protein signals normalized to the corresponding GAPDH levels. Data are expressed as means ± SEM. Statistical analysis was conducted by independent-samples t -test. * P =0.05 versus LID + Tat-D1Rc. Abbreviations: 6-OHDA, 6-hydroxydopamine; l -dopa, L-3,4-dihydroxyphenylalanine; LID, l -dopa-induced dyskinesia; SEM, standard error of mean; Tat-D1Ri, Tat-fusion interfering peptide; Tat-D1Rc, Tat-fusion control peptide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Membranes were blocked in 5% nonfat milk for 1 hour at room temperature and incubated with a rabbit primary antibody against D1R (Millipore) or a mouse primary antibody against GluN1 (Millipore) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Group I mGluR antagonist rescues the deficit of D1-induced LTP in a mouse model of fragile X syndrome

doi: 10.1186/1750-1326-7-24

Figure Lengend Snippet: Rescue of D1-induced LTP by DL-AP3 in the Fmr1 KO mice. (A) The pairing training produced a significant, long-lasting potentiation of synaptic responses in Fmr1 WT mice (n = 12 slices/5 mice), but not in Fmr1 KO mice (n = 9 slices/4 mice); (B) DL-AP3 (10 μM) did not alter the amplitude of LTP in Fmr1 WT mice (n = 9 slices/3 mice). DL-AP3 (10 μM) failed to induce LTP in Fmr1 KO mice (n = 11 slices/5 mice). (C) SKF81297 (5μM) facilitated LTP induction in Fmr1 WT mice (n = 8 slices/3 mice), but failed to induce LTP in Fmr1 KO mice (n = 11 slices/5 mice). (D) Bath application of SKF81297 (5 μM) and DL-AP3 (10 μM) induced LTP in Fmr1 WT mice (n = 10 slices/3 mice) (n = 10 slices/3 mice) and markedly rescued the LTP induction by SKF81297 in the Fmr1 KO mice (n = 12 slices/4 mice). (E) SCH23390 (10 μM) blocked the LTP by synergistic application of SKF81297 and DL-AP3 in the KO mice (n = 13 slices/4 mice). (F and G) SKF81297 (5 μM, 30 min) or DL-AP3 (10 μM, 30 min) had no effect on basal synaptic responses without pairing training (n = 8) in the Fmr1 WT and KO mice. (H) Summary of the effects of DL-AP3 or/and SKF81297 on the LTP induction. * p < 0.05, ** p < 0.01 compared with WT; # p < 0.05 compared with control; && p < 0.01 compared with SKF81297 + DL-AP3 in WT mice; @ p < 0.05 compared with SKF81297 + DL-AP3 in KO mice.

Article Snippet: Equal amounts of protein (50 μg) from the cultures were separated and electrotransferred onto PDVF membranes (Invitrogen), which were probed with antibodies for mGluR5 (1:500, Abcam, Cambridge, MA), GluR1 (1:300, Abcam), NR2A (1:200, Millipore), NR2B (1:1000, Millipore), p-NR2B Tyr1472 (1:1000, Cell signaling), GRK2 (1:1000, Santa Cruz), D1 receptor (1:1000, Millipore), FMRP (1:1000, Millipore), anti-phosphoserine of D1 receptor (1:2500, BD Biosciences), with β-actin (1:10000, Sigma) or cadherin (1:2000, Sigma) as loading control.

Techniques: Produced

Journal: Molecular Neurodegeneration

Article Title: Group I mGluR antagonist rescues the deficit of D1-induced LTP in a mouse model of fragile X syndrome

doi: 10.1186/1750-1326-7-24

Figure Lengend Snippet: Expression of GRK2 and D1 receptor phosphorylation. Expression levels of GRK2 were decreased in cytosol preparation (A) and increased in membrane preparation (B) from the cultures of Fmr1 KO mice as compared with KO neurons. Synergistic addition of DL-AP3 (10 μM) and SKF81297 (5 μM) reversed redistribution of GRK2 between membrane and cytosol in Fmr1 KO neurons. (C) Synergistic addition of DL-AP3 (10 μM) and SKF81297 (5 μM) reduced the increase of D1 receptor phosphorylation at serine sites to a level similar to WT neurons. n = 4 dishes. * p < 0.05, ** p < 0.01 compared between Fmr1 WT and KO neurons; # p < 0.05 compared with KO control.

Article Snippet: Equal amounts of protein (50 μg) from the cultures were separated and electrotransferred onto PDVF membranes (Invitrogen), which were probed with antibodies for mGluR5 (1:500, Abcam, Cambridge, MA), GluR1 (1:300, Abcam), NR2A (1:200, Millipore), NR2B (1:1000, Millipore), p-NR2B Tyr1472 (1:1000, Cell signaling), GRK2 (1:1000, Santa Cruz), D1 receptor (1:1000, Millipore), FMRP (1:1000, Millipore), anti-phosphoserine of D1 receptor (1:2500, BD Biosciences), with β-actin (1:10000, Sigma) or cadherin (1:2000, Sigma) as loading control.

Techniques: Expressing

Journal: Molecular Neurodegeneration

Article Title: Group I mGluR antagonist rescues the deficit of D1-induced LTP in a mouse model of fragile X syndrome

doi: 10.1186/1750-1326-7-24

Figure Lengend Snippet: Expression of D1 receptor, mGluR5, and glutamate receptor subunits. (A) Representatives of western blot from the prefrontal cortex of Fmr1 WT and KO mice. (B) There were no differences in expressions of D1 receptor, mGluR5, NR2A, and NR2B between the WT and Fmr1 KO mice ACC. n = 4 mice per group. (C) Representatives of western blot to detect the level of phosphorylation of NR2B subunit at Tyr-1472 (p-NR2B-Tyr1472). (D) Level of p-NR2B-Tyr1472 was increased after treatment with SKF81297 (5 μM) alone or combining with DL-AP3 (10 μM) in neurons from Fmr1 WT and KO mice. n = 4 dishes. * p < 0.05, ** p < 0.01 compared with control; # p < 0.05 compared between Fmr1 WT and KO mice.

Article Snippet: Equal amounts of protein (50 μg) from the cultures were separated and electrotransferred onto PDVF membranes (Invitrogen), which were probed with antibodies for mGluR5 (1:500, Abcam, Cambridge, MA), GluR1 (1:300, Abcam), NR2A (1:200, Millipore), NR2B (1:1000, Millipore), p-NR2B Tyr1472 (1:1000, Cell signaling), GRK2 (1:1000, Santa Cruz), D1 receptor (1:1000, Millipore), FMRP (1:1000, Millipore), anti-phosphoserine of D1 receptor (1:2500, BD Biosciences), with β-actin (1:10000, Sigma) or cadherin (1:2000, Sigma) as loading control.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Dopamine D2 Receptor Stimulation Potentiates PolyQ-Huntingtin-Induced Mouse Striatal Neuron Dysfunctions via Rho/ROCK-II Activation

doi: 10.1371/journal.pone.0008287

Figure Lengend Snippet: A–D: primary striatal neurons were transfected or not (Control) with Cont siRNA, D1R siRNA and D2R siRNA. After 72 hours, mRNAs were extracted followed by RT-PCR (A and B) and protein expression levels detected by immunoblot (C and D). A: RT-PCR of D1R gene and, B: RT-PCR D2R. Note the presence of both D2R long and short isoforms. For A and B: ARP gene was used as an housekeeping gene. B and D: immunoblot using specific anti-D1R (C), and specific D2R (D) antibodies. β-tubulin was used for control loading.

Article Snippet: After blocking in 5% nonfat dry milk (for ROCK-I and ROCK-II) or 5% BSA (Bovine Serum Albumin, SIGMA) (for D1R and D2R immunobloting) in TBS for 1 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies, rabbit polyclonal anti-ROCK-I anti-ROCK-II (1/500, Santa Cruz), mouse monoclonal anti-D1R (1/250, Millipore), rabbit polyclonal anti-D2R (1/1000, Millipore) and mouse monoclonal anti-β-tubulin (1/5000,Sigma), and revealed with appropriate anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (1/5000, Jackson ImmunoResearch Laboratories) and the enhanced chemiluminescent (ECL) detection system (Pierce).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot