Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: The effects of D1-D2 heteromer stimulation and inactivation on basal conditioned place preference. (A) Vehicle-conditioned rats did not exhibit a preference toward a particular chamber. D1-D2 heteromer stimulation by SKF 83959 (1.5 mg/kg, s.c.) induced conditioned place aversion (CPA) as the animals spent significantly less time in the drug paired chamber. (B) SKF 83959-induced CPA was abolished by pre-treatment by the D1-D2 heteromer selective disrupting peptide, TAT-D1, but not the control TAT-Sc peptide. (C) Inactivation of D1-D2 heteromer by TAT-D1 resulted in conditioned place preference (CPP) as the rats spent significantly more time in the drug paired chamber, not observed with the control TAT-Sc. (D) Representative western blots (inset) and histogram showing the amount of D1R co-immunoprecipitated with D2R from the NAc of rats treated with saline or SKF 83959. Pretreatment with TAT-D1 led to decreased co-immunoprecipitated receptors. An aliquot of each sample was used as a control for WB (input control). (E,F) The CPA induced by D1-D2 heteromer stimulation was abolished by Cdk5 inhibitor roscovitine pre-treatment (200 nmol, i.c.v, E ) or intra-accumbal injection (30 nmol, F ). (G) Representative western blot and histogram showing the density of Thr75-DARPP-32 phosphorylation (pT75) relative to GAPDH (as loading control). Data represent means ± SEM of n = 8–10 rats/group. ( * p < 0.05, ** p < 0.01: compared to saline).

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Conditioned Place Preference, Western Blot, Immunoprecipitation, Injection

Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: Activation of Thr75-DARRP-32 by D1-D2 heteromer in rat NAc. (A,B) Rats ( n = 8/group) were injected with saline or SKF 83959 (1.5 mg/kg, s.c.), sacrificed 15, 45, or 90 min later, and phospho-Thr34-DARPP-32 (pT34) or phospho-Thr75-DARPP-32 (pT75) analyzed by western blot with GAPDH as loading control. (A) Representative blots of pT34 and pT75. (B) Quantification of blots from all animals represented as % mean ± SEM of control (saline values), ( ** p < 0.05). (C–F) Rats were injected with saline or SKF 83959 (1.5 mg/kg), sacrificed 15 min later, and immunohistochemistry performed using anti-pT34 or anti-pT75 assessed in the three types of MSNs: D1R-only (red arrow), Enk-only (D2R, green arrow) or D1R and ENK (D1-D2 heteromer)-expressing neurons (yellow arrow). (C) Representative confocal images of pT75-DARPP-32. (D) Quantification of pT75-DARPP-32 fluorescence in MSNs ( n = numbers of neurons from at least N = 3 rats/condition). (E) Representative confocal images of pT34-DARPP-32. (F) Quantification of pT34 fluorescence in MSNs. Data represents means ± SEM after removal of non-specific background. ( ** p < 0.01).

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Activation Assay, Injection, Western Blot, Immunohistochemistry, Expressing, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p < 0.05 and ** p < 0.01 represent significant differences from control. (C) Representative confocal images of pERK assessed in the three types of MSNs: D1R-only (red arrow), Enk-only (D2R, green arrow) or D1R and ENK (D1-D2 heteromer)-expressing neurons (yellow arrow) in MSNs from NAc of saline- or SKF 83959-treated rats. (D) Quantification of pERK fluorescence in MSNs in the NAc. Results are the mean ± SEM of data after removing the non-specific background ( n = number of MSNs from N = at least 3 rats/condition). ( ** p < 0.001; *** p < 0.0001). (E) Representative immunohistochemistry images and their quantification obtained using an antibody against ΔFosB and a secondary antibody conjugated to Alexa-488. Nuclei are stained by DAPI. Rats were treated for 7 days with cocaine (10 mg/kg, i.p.) without or with co-injection of SKF 83959 (1 mg/kg, s.c.). Disrupting the heteromer by repeated injections of TAT-D1 had the same effect as repeated injections of cocaine. Results are means ± SD obtained by the analysis of n = 1,500–1,700 neurons from the NAc of N = 3 rats/condition.

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Western Blot, Injection, Expressing, Fluorescence, Immunohistochemistry, Staining

Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: Evidence for the existence of dopamine D1-D2 receptor heteromer in rat NAc. (A) Proximity ligation assay (PLA) was used to visualize and detect D1R and D2R close proximity. (A1) A scheme depicts the PLA probes used in the present study. (A2–A4) Representative images of PLA signals (red dots) in neurons (nuclei stained by DAPI) in rat caudate putamen (CPu), nucleus accumbens core (NAc-core) and shell (NAc-shell) subregions. (A5) Graph representing the percent of neurons with a positive PLA signal. (A6) Representative image of PLA signals in neurons (nuclei stained by DAPI) in rat NAc-core using the second set of antibodies. (B) Representative images of immunohistochemistry using D1R antibody (D1R-Ab) or D2R antibody (D2R-Ab) directly conjugated to Alexa-488 or Alexa-568, respectively, in the NAc-shell. Direct confocal FRET analysis was performed, reflected by FRET efficiency (FRET E) and the distance between the dipoles, less than 10 nm (100 Å). (C) A representative close-up of a single MSN cell body from NAc showing D1R-D2R colocalization (left), D1-D2 heteromer FRET efficiency (center) and relative distance between receptors (right). (D,E) Histograms showing FRET E ratios (D) and distance (E) obtained from MSN cell bodies from NAc ( n = 24). Bars are 10 μm.

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Proximity Ligation Assay, Staining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: Leukemic progenitor assays replicate patterns of patient response to DRD2 antagonist TDZ (A) Leukemic blast counts were monitored before and after treatment with TDZ as a monotherapy in 11 relapsed or refractory AML patients (NCT02096289). Percentage change in blasts in the peripheral blood on day 5 versus day 1 is reported after treatment with TDZ. Percentage change in BM blast content is reported for trial patient 2T and 9T in the absence of circulating blast values. Partial response and progressive disease patterns are indicated as “response” and “no response” and are illustrated as gray versus black silhouettes, respectively. (B) Candidate trial patient samples from either response group were interrogated for progenitor content at baseline (day 1) and after clinical exposure to TDZ (day 5) using limiting dilution analysis (LDA). Leukemic progenitor frequency was estimated by LDA analysis and normalized to day 1. Baseline progenitor frequency of 1 in 75,000 cells was considered the progenitor frequency for trial patient 3T at day 1 since an absolute frequency was not achieved with the analysis of 75,000 cells for this patient. Dashed lines represent 95% confidence interval. Raw colony counts are shown in Figure S1 D. (C) Trial patient samples obtained at baseline were exposed to TDZ (“+TDZ”) versus DMSO control (“−TDZ”) for 24 h, followed by analysis of progenitor cell function in CFU assays. Data are normalized to DMSO control. Before normalization, the average DMSO control values were 79 and 2 colonies for trial patients 1T and 8T (non-responders) and 61, 28, 56, 2, 11, 28, and 14 colonies for trial patients 2T, 4T, 6T, 7T, 9T, 10T, and 11T, respectively (responders). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (D) Correlation between percentage change in leukemic blast levels versus percentage change in progenitor capacity (demonstrated in C). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (E) Schematic illustrating in vivo AML xenografts were treated with TDZ (22.5 mg/kg “+”) or 30% captisol (vehicle control “−”) in vivo , followed by analysis of leukemic chimerism levels (F), gene expression analysis (G), and progenitor CFU assays (H). (F) Leukemic chimerism levels (hCD45 + CD33 + ) after in vivo treatment with TDZ relative to vehicle control (“−“). Symbols represent individual recipient mice. ∗p = 0.05 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. (G) Gene set enrichment analysis (GSEA) plot of a gene set representing cellular pathways associated with AML (Kyoto Encyclopedia of Genes and Genomes [KEGG]; ), applied to transcription profiles from TDZ-treated versus vehicle control-treated AML xenografts derived from AMLs 1, 3, and 4. (H) Human AML grafts were recovered from mouse BM and evaluated in progenitor CFU assays. Symbols represent individual CFU wells, plated using cells recovered from a minimum of 2 individual mice per condition. Colony-forming capacity for AML 4 was not detectable with up to 150,000 human cells assayed. ∗∗∗p ≤ 0.0001 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. Data are summarized as means ± SEMs. See also and and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Control, Cell Function Assay, In Vivo, Gene Expression, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: DRD2 expression profiles reliably predict functional response to DRD antagonism (A) DRD2 expression patterns within leukemic CD34 + cells. Dotted line represents FMO control (left). Comparison of DRD2 protein levels in CD34 + cells of AML patient versus healthy donor samples (right). Healthy donor samples consist of cord blood (n = 3), adult mobilized peripheral blood (n = 3), and adult non-mobilized peripheral blood (n = 5). Blue versus red shading indicates the threshold of normal versus aberrant DRD2 levels. ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test). (B) DRD2 protein expression within CD34 + subset of low versus intermediate-/high-risk AML patients based on ELN criteria. Dots represent individual AML patients. ∗∗p = 0.006 (Mann-Whitney U test). (C) Mononuclear cells (MNCs) isolated from healthy donors and AML patients were treated with TDZ or DMSO (vehicle control, “−”) for 24 h and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n = 3–10 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). Source data can be found in . (D) Proliferative capacity of leukemic versus healthy progenitor units was compared after in vitro exposure to TDZ for 24 h. Cell number output per colony was evaluated by custom scripts as a measure of proliferation. (E) Representative FACS plots demonstrate gating strategy to purify DRD2 + vs DRD2 − human AML cells (left) and human leukemic chimerism in mice transplanted with 1 million DRD2 + or DRD2 − human AML cells. (F) Western blot of DRD2, activated CREB (p-CREB at Ser-133), and histone H3 (loading control) in DRD2 + versus DRD2 − sorted fractions illustrated in (E). (G) Representative whole-well CFU images after treatment with dopamine (DA) at physiological levels (10 nM) versus DMSO control (-DA). (H) Progenitor cell activity was quantified in n = 6 distinct AML patients after treatment with physiological levels of DA (10–100 nM) relative to DMSO control. n = 2–3 CFU wells per AML sample. ∗p = 0.03 (unpaired t test). (I) Circulating DA levels in healthy individuals (n = 8 healthy adult peripheral blood (PB) and 11 cord blood (CB) samples, as hollow circles and squares, respectively) versus n = 11 AML patients (black circles). ∗p = 0.04 (unpaired t test). Data are summarized as means ± SEMs relative to vehicle control. See also Figure S3 and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Expressing, Functional Assay, Control, Comparison, MANN-WHITNEY, Isolation, In Vitro, Western Blot, Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: TDZ + displays superior potency and reduced toxicity relative to TDZ (A) Chiral separation of TDZ using supercritical fluid chromatography. Chromatograms show the first and second peaks, indicating the (−) enantiomer “TDZ − ” and (+) enantiomer “TDZ + ,” respectively. Purified enantiomers were evaluated for effects on cAMP levels (B), and in progenitor CFU assays (C and D). (B) cAMP levels were evaluated after in vitro treatment with TDZ and its two enantiomers in AML cell lines (NB4 and OCI-AML3) and primary patient cells (AMLs 2, 9, and 27). Symbols represent individual CFU wells. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 (unpaired t test). (C) AML patient cells were exposed to TDZ and its 2 enantiomers for 24 h in a dose-response assay in vitro , and subsequently evaluated in progenitor CFU assays. Bar graphs summarize half-maximal inhibitory concentration (IC 50 ) in progenitor CFU assays performed with AML patient cells. ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001 (paired t test). (D) Comparison of TDZ and TDZ + IC 50 for individual AML patients in CFU assays (represented in C). ∗∗p = 0.004 (paired t test). (E) A 30-min monitoring of QTc level changes after intravenous injection of TDZ and TDZ + in a guinea pig assay (n = 5 animals per cohort). QTc increases over 5% were considered indicators of safety risks. No group averages were statistically different from baseline values (repeated-measures ANOVAs). (F) DRD2 transcript (Gene: 1813) was analyzed from TGCA (tumor and normal tissue) and GTEx (normal tissue) RNA-sequencing projects. Data points represent normalized gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) for DRD2 from individual cancer patients or healthy donors. ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test), ∗∗p = 0.01 (Kolmogorov-Smirnov test). Data are summarized as means ± SEMs. See also Figure S5 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Supercritical Fluid Chromatography, Purification, In Vitro, Concentration Assay, Comparison, Injection, RNA Sequencing, Gene Expression, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging

Journal: Reproductive biomedicine online

Article Title: Social psychogenic stress promotes the development of endometriosis in mouse.

doi: 10.1016/j.rbmo.2016.11.012

Figure Lengend Snippet: Figure 4 – Immmunoreactivity staining of different markers in ectopic lesions in different groups. (A) Representative immunostaining of ADRB2 in endometrium in CONTROL and SHAM mice and in ectopic endometrium in STRESSED and UNSTRESSED mice. ADRB2 and DRD2 immunoreactivity was both seen primarily in glandular epithelial cells and was localized in the cytoplasm. Scale bar = 125 μm. (B) Representative immunostaining of DRD2, VEGF, CD31, CD41, F4/80, PCNA and α-SMA in the ectopic lesions in UNSTRESSED and STRESSED groups. VEGF immunoreactivity was seen primarily in glandular epithelial cells and was localized in the cytoplasm. CD31 immunostainings were seen mostly in vascular endothelial cells. CD41 shows the extent of platelet aggregation and F4/80 immunoreactivity represents the extent of macrophage infiltration. PCNA immunoreactivity was seen both in glandular epithelial cells and stromal cells were localized in the cell nucleus, but the change of immunoreactivity in glandular epithelial cells was more obvious. α-SMA staining were seen mostly in the stromal component of the ectopic lesions. Scale bar = 125 μm. Mice in stressed (STRS) and unstressed (UNSTRS) groups had undergone endometriosis-inducing surgery, while mice the SHAM group underwent non-endometriosis-inducing surgery. Mice in unstressed (UNSTRS), SHAM and control (CTL) groups were not exposed to stress.

Article Snippet: For negative controls, the immunoglobulin G (IgG) from the rabbit serum (Sigma, Darmstadt, Germany) was used instead of primary antibodies against ADRB2, DRD2, VEGF, CD31, CD41, α-SMA and PCNA except for F4/80, for which the IgG was from the rat serum (Boster, Wuhan, China) was used, all used at the same concentration with its corresponding primary antibody.

Techniques: Staining, Immunostaining, Control

Journal: Translational Psychiatry

Article Title: Human umbilical cord-derived mesenchymal stem cells alleviate schizophrenia-relevant behaviors in amphetamine-sensitized mice by inhibiting neuroinflammation

doi: 10.1038/s41398-020-0802-1

Figure Lengend Snippet: a Intravenous human UC-MSC was infused intravenously 1 day following the final 3XAMP injection after which FST, SI, and LI were performed. Mice were sacrificed at 35 days following hUC-MSC treatment. In behavioral assessments, hUC-MSC did not restore reduced immobility time in FST ( b ) and social deficit in SI ( c ), but rescued disruption of LI ( d ) in 3XAMP treated mice. n = 10 in FST and SI, LI test was performed three times independently, n = 27–30 per group. In another amphetamine treatment regimen (escalating regimen) ( e ), Sociality was impaired ( f ) and PPI was not changed in all groups ( g ) hUC-MSC reduced immobility time in TST ( h ) and FST ( i ) in amphetamine-sensitized mice by escalating regimen. TST and FST was performed twice independently n = 19–20 per group. j Tyrosine hydroxylase (TH), dopamine 2 receptor (D2R), dopamine transporter (DAT), glutamate decarboxylase 67 (GAD67), PSD95, and synpatophysin were assessed by western blot analysis and their expression was quantified using Image J in 3XAMP regimen mice, with n = 4 per group. k An immunofluorescence study was performed to determine the effect of hUC-MSC on number of Iba-1 positive cells in the ventral region of the striatum (vSt) and the medial prefrontal cortex (mPFC) and was quantified using Image J in in the 3XAMP regimen mice; n = 3 per group. Data are shown as mean ± standard error of the mean (SEM); scale bar is 50 μm. * p < 0.05, ** p < 0.01. *** p < 0.001 compared with the CON or NPE. # p < 0.05 compared with the AMP.

Article Snippet: Primary antibodies were dissolved in the blocking buffer and the membranes were immunoblotted with anti- bodies against Dopamine 2 receptor (D2R 1:200, Bioss #bs-1008R), dopamine transporter (DAT, 1:200, abcam#ab111468), tyrosine hydroxylase (1:200, abcam#ab112), glutamate decarboxylase 67 (GAD67, 1:500, #ab26116), PSD95 (1:300, #ab18258), synaptophysin (1:500, #ab14692) and beta-actin (1:1000, Cell Signaling).

Techniques: Injection, Western Blot, Expressing, Immunofluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Effects of Chemogenetic Inhibition of D1 or D2 Receptor-Containing Neurons of the Substantia Nigra and Striatum in Mice With Tourette Syndrome

doi: 10.3389/fnmol.2021.779436

Figure Lengend Snippet: Experimental design. (A) Schematic diagram of the virus injection area. (B) Experimental design timeline. A total of 336 mice [including 144 D1R-cre mice and 24 wild-type littermate (WT) mice, 144 D2R-cre mice, and 24 WT mice] participated in this experiment. After adaptive feeding for 1 week, they were randomly divided into four large groups (84 mice in each large group), among which 72 D1R-cre mice and 12 WT mice were further divided into two small groups, and 72 D2R-cre mice and 12 WT littermate mice were further divided into two small groups. On the first day, one small group of D1R-cre mice and WT littermate mice, one small group of D2R-cre mice and WT mice were injected with the virus in the bilateral dSTR (200 nL/side), the other two small groups were injected with the virus in the bilateral SNpc (200 nL/side). Eighty-four mice in each large group were randomly divided into 14 groups according to the injections of different drugs or virus, including WT + Vehicle + mCherry + i.p. Saline (group a), D1R or D2R-cre + Vehicle + mCherry + i.p. Saline group (group b), D1R or D2R-cre + Vehicle + Gq + i.p. Saline group (group c), D1R or D2R-cre + Vehicle + Gi + i.p. Saline group (group d), D1R or D2R-cre + IDPN + mCherry + i.p. Saline group (group e), D1R or D2R-cre + IDPN + Gq + i.p. Saline group (group f), D1R or D2R-cre + IDPN + Gi + i.p. Saline group (group g), WT + Vehicle + mCherry + i.p. CNO group (group h), D1R or D2R-cre + Vehicle + mCherry + i.p. CNO group (group i), D1R or D2R-cre + Vehicle + Gq + i.p. CNO group (group j), D1R or D2R-cre + Vehicle + Gi + i.p. CNO group (group k), D1R or D2R-cre + IDPN + mCherry + i.p. CNO group (group l), D1R or D2R-cre + IDPN + Gq + i.p. CNO group (group m) and D1R or D2R-cre + IDPN + Gi + i.p. CNO group (group n) ( n = 6 mice in each group). Mice in the groups a, b, e, h, i, and l were injected with a control virus (mCherry). Mice in the groups c, f, j, and m were injected with hM3Dq. Mice in group d, g, k, and n were injected with hM4Di. After the virus injection, all mice were back to the cage to rest for 4 days (days 2–5). On days 6–12, mice in groups e, f, g, l, m, and n were intraperitoneally injected with IDPN at 10:00 a.m. once daily, and mice in the other groups were intraperitoneally injected with 0.9% saline (Vehicle, contrast with IDPN). On the 23rd day, mice in the a–g groups were intraperitoneally injected with 0.9% saline (contrast with CNO), and mice in the h–n groups were intraperitoneally injected with clozapine N-oxide (CNO, Sigma, St. Louis, MO, United States, 1 mg/kg). Thirty minutes later, behavioral tests were carried out to compare the differences between the groups. Brain tissues for immunofluorescence labeled were collected on day 24.

Article Snippet: The sections were rinsed in 0.01 M PBS, blocked for 1 h with 5% donkey serum and 0.2% Tween-20 in PBS, and then incubated with the following primary antibodies at 37°C for 1 h and at 4°C overnight: rabbit anti-D1R (1:500, Ab20066, Abcam) and rabbit anti-D2R (1:100, PAA673Mu01, Cloud-Clone).

Techniques: Injection, Immunofluorescence, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Effects of Chemogenetic Inhibition of D1 or D2 Receptor-Containing Neurons of the Substantia Nigra and Striatum in Mice With Tourette Syndrome

doi: 10.3389/fnmol.2021.779436

Figure Lengend Snippet: Effects of activation or inhibition of the D2R-containing neurons in the SNpc and dSTR on stereotyped behavior in mice. (A) Evaluations of stereotyped behavior scores of mice injected with virus in the SNpc 30 min after the injection of saline (i.p. Saline) in the WT + Vehicle + mCherry + i.p. Saline group (WT + m), D2R-cre + Vehicle + mCherry + i.p. Saline group (Veh + m), D2R-cre + IDPN + mCherry + i.p. Saline group (TS + m), D2R-cre + Vehicle + Gq + i.p. Saline group (Veh + Gq), D2R-cre + IDPN + Gq + i.p. Saline group (TS + Gq), D2R-cre + Vehicle + Gi + i.p. Saline group (Veh + Gi), D2R-cre + IDPN + Gi + i.p. Saline group (TS + Gi) on day 23. (B) Evaluations of stereotyped behavior scores of mice injected with virus in the SNpc 30 min after the injection of CNO (i.p. CNO) in the WT + Vehicle + mCherry + i.p. CNO group (WT + m), D2R-cre + Vehicle + mCherry + i.p. CNO group (Veh + m), D2R-cre + IDPN + mCherry + i.p. CNO group (TS + m), D2R-cre + Vehicle + Gq + i.p. CNO group (Veh + Gq), D2R-cre + IDPN + Gq + i.p. CNO group (TS + Gq), D2R-cre + Vehicle + Gi + i.p. CNO group (Veh + Gi), D2R-cre + IDPN + Gi + i.p. CNO group (TS + Gi) on day 23. (C) Evaluations of stereotyped behavior scores of mice injected with virus in the dSTR 30 min after the injection of saline (i.p. Saline) in each group on day 23. (D) Evaluations of stereotyped behavior scores of mice injected with virus in the dSTR 30 min after the injection of CNO (i.p. CNO) in each group on day 23. Data are expressed as mean ± SEM ( n = 6 mice in each group), and the black line segment indicates the differences between groups, * represents P < 0.05 between marked groups, n.s. represents P > 0.05 between marked groups.

Article Snippet: The sections were rinsed in 0.01 M PBS, blocked for 1 h with 5% donkey serum and 0.2% Tween-20 in PBS, and then incubated with the following primary antibodies at 37°C for 1 h and at 4°C overnight: rabbit anti-D1R (1:500, Ab20066, Abcam) and rabbit anti-D2R (1:100, PAA673Mu01, Cloud-Clone).

Techniques: Activation Assay, Inhibition, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Effects of Chemogenetic Inhibition of D1 or D2 Receptor-Containing Neurons of the Substantia Nigra and Striatum in Mice With Tourette Syndrome

doi: 10.3389/fnmol.2021.779436

Figure Lengend Snippet: Effects of activation or inhibition of D2R-containing neurons in the SNpc on total distance and resting time in the open-field test in mice. (A) Evaluations of the trajectory in the open-field test in mice 30 min after the injection of saline (i.p. Saline) or CNO (i.p. CNO) in the WT + Vehicle + mCherry + i.p. Saline group (WT + m), D2R-cre + Vehicle + mCherry + i.p. Saline group (Veh + m), D2R-cre + IDPN + mCherry + i.p. Saline group (TS + m), D2R-cre + Vehicle + Gq + i.p. Saline group (Veh + Gq), D2R-cre + IDPN + Gq + i.p. Saline group (TS + Gq), D2R-cre + Vehicle + Gi + i.p. Saline group (Veh + Gi), D2R-cre + IDPN + Gi + i.p. Saline group (TS + Gi), WT + Vehicle + mCherry + i.p. CNO group (WT + m), D2R-cre + Vehicle + mCherry + i.p. CNO group (Veh + m), D2R-cre + IDPN + mCherry + i.p. CNO group (TS + m), D2R-cre + Vehicle + Gq + i.p. CNO group (Veh + Gq), D2R-cre + IDPN + Gq + i.p. CNO group (TS + Gq), D2R-cre + Vehicle + Gi + i.p. CNO group (Veh + Gi), D2R-cre + IDPN + Gi + i.p. CNO group (TS + Gi) on day 23. (B) Total distance in the open-field test within 10 min in mice of each group 30 min after the injection of saline (i.p. Saline) on day 23. (C) Total distance in the open-field test within 10 min in mice of each group 30 min after the injection of CNO (i.p. CNO) on day 23. (D) Resting time in the open-field test within 10 min in mice of each group 30 min after the injection of saline (i.p. Saline) on day 23. (E) Resting time in the open-field test within 10 min in mice of each group 30 min after the injection of CNO (i.p. CNO) on day 23. Data are expressed as mean ± SEM ( n = 6 mice in each group), and the black line segment indicates the differences between groups, * represents P < 0.05 between marked groups, and n.s. represents P > 0.05 between marked groups.

Article Snippet: The sections were rinsed in 0.01 M PBS, blocked for 1 h with 5% donkey serum and 0.2% Tween-20 in PBS, and then incubated with the following primary antibodies at 37°C for 1 h and at 4°C overnight: rabbit anti-D1R (1:500, Ab20066, Abcam) and rabbit anti-D2R (1:100, PAA673Mu01, Cloud-Clone).

Techniques: Activation Assay, Inhibition, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Effects of Chemogenetic Inhibition of D1 or D2 Receptor-Containing Neurons of the Substantia Nigra and Striatum in Mice With Tourette Syndrome

doi: 10.3389/fnmol.2021.779436

Figure Lengend Snippet: Effects of activation or inhibition of D2R-containing neurons in the dSTR on total distance and resting time in the open-field test in mice. (A) Evaluations of the trajectory in the open-field test in mice 30 min after the injection of saline (i.p. Saline) or CNO (i.p. CNO) in the WT + Vehicle + mCherry + i.p. Saline group (WT + m), D2R-cre + Vehicle + mCherry + i.p. Saline group (Veh + m), D2R-cre + IDPN + mCherry + i.p. Saline group (TS + m), D2R-cre + Vehicle + Gq + i.p. Saline group (Veh + Gq), D2R-cre + IDPN + Gq + i.p. Saline group (TS + Gq), D2R-cre + Vehicle + Gi + i.p. Saline group (Veh + Gi), D2R-cre + IDPN + Gi + i.p. Saline group (TS + Gi), WT + Vehicle + mCherry + i.p. CNO group (WT + m), D2R-cre + Vehicle + mCherry + i.p. CNO group (Veh + m), D2R-cre + IDPN + mCherry + i.p. CNO group (TS + m), D2R-cre + Vehicle + Gq + i.p. CNO group (Veh + Gq), D2R-cre + IDPN + Gq + i.p. CNO group (TS + Gq), D2R-cre + Vehicle + Gi + i.p. CNO group (Veh + Gi), D2R-cre + IDPN + Gi + i.p. CNO group (TS + Gi) on day 23. (B) Total distance in the open-field test within 10 min in mice of each group 30 min after the injection of saline (i.p. Saline) on day 23. (C) Total distance in the open-field test within 10 min in mice of each group 30 min after the injection of CNO (i.p. CNO) on day 23. (D) Resting time in the open-field test within 10 min in mice of each group 30 min after the injection of saline (i.p. Saline) on day 23. (E) Resting time in the open-field test within 10 min in mice of each group 30 min after the injection of CNO (i.p. CNO) on day 23. Data are expressed as mean ± SEM ( n = 6 mice in each group), and the black line segment indicates the differences between groups, * represents P < 0.05 between marked groups, and n.s. represents P > 0.05 between marked groups.

Article Snippet: The sections were rinsed in 0.01 M PBS, blocked for 1 h with 5% donkey serum and 0.2% Tween-20 in PBS, and then incubated with the following primary antibodies at 37°C for 1 h and at 4°C overnight: rabbit anti-D1R (1:500, Ab20066, Abcam) and rabbit anti-D2R (1:100, PAA673Mu01, Cloud-Clone).

Techniques: Activation Assay, Inhibition, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Effects of Chemogenetic Inhibition of D1 or D2 Receptor-Containing Neurons of the Substantia Nigra and Striatum in Mice With Tourette Syndrome

doi: 10.3389/fnmol.2021.779436

Figure Lengend Snippet: Hypothesis diagram of experimental mechanism. (A) Hypothesis diagram of chemogenetic inhibition or activation of D1R-containing neurons in the SNpc. (B) Hypothesis diagram of chemogenetic inhibition or activation of D2R-containing neurons in the SNpc. (C) Hypothesis diagram of chemogenetic inhibition or activation of D1R-containing neurons in the dSTR. (D) Hypothesis diagram of chemogenetic inhibition or activation of D2R-containing neurons in the dSTR. The solid line or plus sign indicates activation. The dashed line or minus sign indicates inhibition.

Article Snippet: The sections were rinsed in 0.01 M PBS, blocked for 1 h with 5% donkey serum and 0.2% Tween-20 in PBS, and then incubated with the following primary antibodies at 37°C for 1 h and at 4°C overnight: rabbit anti-D1R (1:500, Ab20066, Abcam) and rabbit anti-D2R (1:100, PAA673Mu01, Cloud-Clone).

Techniques: Inhibition, Activation Assay