Journal: Experimental and Therapeutic Medicine

Article Title: Natural emodin reduces myocardial ischemia/reperfusion injury by modulating the RUNX1/miR‑142‑3p/DRD2 pathway and attenuating inflammation

doi: 10.3892/etm.2022.11681

Figure Lengend Snippet: miR-142-3p negatively regulates the expression of DRD2. (A) Reverse transcription-quantitative PCR for the identification of the downstream target gene of miR-142-3p by overexpressing miR-142-3p mimic in H9c2 cells. n=4. (B) The DRD2 protein levels were detected by western blotting in cardiac tissues with MIRI or miR-142-3p antagomir (anti-ago) treatment. (C) Summary for the protein expression of DRD2 based on (B), n=3. (D) DRD2 protein levels were detected by western blotting in H9c2 cells with H/R injury or emodin treatment. (E) Summary for the protein expression of DRD2 based on (D), n=3. (F) DRD2 protein levels were measured using western blotting in cardiac tissues with MIRI or emodin treatment. Three bands represent three independent samples. (G) Summary for the protein expression of DRD2 based on (F), n=3. * P<0.05, ** P<0.01. NC, negative control; MIRI, myocardial ischemia/reperfusion injury; anti-ago, antagomir; DRD2, dopamine receptor D2; Ctrl, control; miR, microRNA; H/R, hypoxia/reoxygenation.

Article Snippet: After blocking in TBST (0.1% Tween-20) containing 5% fat-free milk at room temperature for 1 h, the membrane was incubated with primary anti-RUNX1 (1:1,000; cat. no. ab229482; Abcam), anti-DRD2 (1:500; cat. no. bs-20730R; BIOSS), anti-phosphorylated (p-)P65 subunit of NF-κB (1:1,000; cat. no. ab76302; Abcam), anti-NF-κB P65 subunit (1:1,000; cat. no. bs-20355R; BIOSS), anti-TNF-α (1:500; cat. no. bs-2081R; BIOSS), anti-IL-6 (1:500; cat. no. ab259341; Abcam) or anti-β-tubulin (1:1,000; cat. no. ab18207; Abcam) antibodies respectively, overnight at 4˚C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Natural emodin reduces myocardial ischemia/reperfusion injury by modulating the RUNX1/miR‑142‑3p/DRD2 pathway and attenuating inflammation

doi: 10.3892/etm.2022.11681

Figure Lengend Snippet: RUNX1/miR-142-3p/DRD2 pathway regulates the NF-κB-dependent inflammatory responses in H9c2 cells with H/R injury. (A) The NF-κB dependent inflammatory response was regulated by overexpression of RUNX1, miR-142-3p mimic and DRD2 in H9c2 cells with H/R injury and detected using western blotting. (B) Bar diagram showing the protein expression levels of RUNX1, DRD2, P65, TNF-α and IL-6 based on (A). n=3. (C) Bar diagram showing the protein expression of functional p-P65 subunit of NF-κB based on (A). n=3. * P<0.05, ** P<0.01. H/R, hypoxia/reoxygenation; DRD2, dopamine receptor D2; p-, phosphorylated-; miR, microRNA; Ctrl, control.

Article Snippet: After blocking in TBST (0.1% Tween-20) containing 5% fat-free milk at room temperature for 1 h, the membrane was incubated with primary anti-RUNX1 (1:1,000; cat. no. ab229482; Abcam), anti-DRD2 (1:500; cat. no. bs-20730R; BIOSS), anti-phosphorylated (p-)P65 subunit of NF-κB (1:1,000; cat. no. ab76302; Abcam), anti-NF-κB P65 subunit (1:1,000; cat. no. bs-20355R; BIOSS), anti-TNF-α (1:500; cat. no. bs-2081R; BIOSS), anti-IL-6 (1:500; cat. no. ab259341; Abcam) or anti-β-tubulin (1:1,000; cat. no. ab18207; Abcam) antibodies respectively, overnight at 4˚C.

Techniques: Over Expression, Western Blot, Expressing, Functional Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Natural emodin reduces myocardial ischemia/reperfusion injury by modulating the RUNX1/miR‑142‑3p/DRD2 pathway and attenuating inflammation

doi: 10.3892/etm.2022.11681

Figure Lengend Snippet: Inhibition of miR-142-3p or overexpression of DRD2 suppresses NF-κB-mediated inflammation in MIRI model of mice. The secretions of inflammatory cytokines (A) IL-6 and (B) TNF-α in cardiac tissues with MIRI of mice were measured by ELISA under the conditions of pretreatment with miR-142-3p antagomir (anti-ago) or overexpression of AAV-DRD2. n=4. (C) P65 and p-P65 were detected using western blotting to determine the activity of NF-κB. (D) Bar diagram showing the protein expression of p-P65 based on (C). n=3. (E) The schematic model for the anti-inflammation action of emodin in MIRI model of mice. ** P<0.01. DRD2, dopamine receptor D2; MIRI, myocardial ischemia/reperfusion injury; anti-ago, antagomir; AAV, adeno-associated virus; p-, phosphorylated-; Ctrl, control; miR, microRNA.

Article Snippet: After blocking in TBST (0.1% Tween-20) containing 5% fat-free milk at room temperature for 1 h, the membrane was incubated with primary anti-RUNX1 (1:1,000; cat. no. ab229482; Abcam), anti-DRD2 (1:500; cat. no. bs-20730R; BIOSS), anti-phosphorylated (p-)P65 subunit of NF-κB (1:1,000; cat. no. ab76302; Abcam), anti-NF-κB P65 subunit (1:1,000; cat. no. bs-20355R; BIOSS), anti-TNF-α (1:500; cat. no. bs-2081R; BIOSS), anti-IL-6 (1:500; cat. no. ab259341; Abcam) or anti-β-tubulin (1:1,000; cat. no. ab18207; Abcam) antibodies respectively, overnight at 4˚C.

Techniques: Inhibition, Over Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Schematic representation of a coronal section of the mouse brain. The blue region defines the WT striatum, in which the Slc1a1 gene encoding EAAC1 is abundantly expressed. (B) Left: Color map representation of the Slc1a1 levels in striatal samples from WT (n=5) and EAAC1 −/− mice (n=5), measured in qRT-PCR experiments. Each sample was run in triplicates (x-axis). Right: Relative amount of the Slc1a1 gene with respect to the Hprt housekeeping gene in WT and EAAC1 −/− mice, measured as 2 -ΔCT (**p=1.8e-3). (C) Fold change in Slc1a1 expression in EAAC1 −/− versus WT mice (2 -ΔCT ). Negligible levels of Slc1a1 (***p=1.5e-7) are detected in EAAC1 −/− mice. (D) Left: Same experiments as described in (B, left), performed on samples from the DLS or VMS of WT mice (n=7). Right: As in (B, right), for samples from the DLS and VMC. No significant difference was detected in the expression levels of Slc1a1 between the WT DLS and VMS (p=0.42). (E) Schematic representation of a coronal section of the mouse brain in which the striatum, from which we extracted the mRNA, is highlighted in yellow. The lighter shade of yellow indicates lower Drd1a gene expression levels in EAAC1 −/− mice. (F) Left: color map representation of the Drdla and Drd2 levels in WT (n=6) and EAAC1 −/− mice (n=10), measured in qRT-PCR experiments. Each sample was run in triplicates (x-axis). Right: relative amount of the Drd1a and Drd2 gene with respect to the Hprt housekeeping gene in WT and EAAC1 −/− mice, measured as 2 -ΔCT . Higher levels of Drd2 are detected in WT (*p=0.011) and EAAC1 −/− mice (***p=9.6e-6). ( G ) Fold change in Drd1a and Drd2 expression in EAAC1 −/− vs WT mice (2 -ΔCT ). Reduced levels of Drd1a (**p=5.0e-3), not Drd2 (p=0.48), are detected in EAAC1 −/− mice. (H-J) As in (E-G), on samples from the DLS and VMS of WT (n=7) and EAAC1 −/− mice (n=6). Lower levels of Drd1a were detected in the VMS of WT (*p=0.018) and EAAC1 −/− mice (***p_7 le_4). In the VMS, there was a significant reduction in the level of Drd1a (*p=0.012). A significant WT-fold change inDrd1a was detected in the DLS (*p=0.037) and VMS (**p=1.6e-3) of EAAC1 −/− mice. (K-M) As in (E-GF), for Drd2 . The level of Drd2 was lower in the VMS compared to the DLS, in WT (n=7, **p=3.4e-3) and EAAC1 −/− mice (n=6, **p=5.1e-3). No significant WT-fold change was detected in the expression level of Drd2 in the DLS and VMS of EAAC1 −/− mice.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Western blots for D1R, D2R and ß-actin in WT (n=ll) and EAAC1 −/− mice (n=8) show decreased levels of D1R (***p=2.7e-4), not D2R (p=0.51), in EAAC1 −/− mice. (B) Western blot analysis for DARPP-32, showing no significant difference in its expression between WT (n=15) and EAAC1 −/− mice (n=12, p=0.92). (C) Western blot analysis for pDARPP-32 T34 (WT (n=10), EAAC1 −/− (n=10), p=0.41), pDARPP-32 175 (WT (n=13), EAAC1 −/− (n=14), p=0.84), pDARPP-32 S97 (WT (n=13), EAAC1 −/− (n=13), p=0.98), pDARPP-32 S130 (WT (n=14), EAAC1 −/− (n=17), *p=0.024). (D) Pie chart representation of the phosphorylation distribution on the T34, T75, S97 and S130 sites of DARPP-32. The red curve highlights the S130 site, which shows increased phosphorylation in EAAC1 −/− mice. (E) As in (C), following data normalization by the average band intensity values measured in WT mice (pDARPP-32 T34 WT (n=10), EAAC1 −/− (n=10), p=0.46; pDARPP-32 T75 WT (n=13), EAAC1 −/− (n=14), p=0.72; pDARPP-32 S97 WT (n=13), EAAC1 −/− (n=13), p=0.89; pDARPP-32 5130 WT (n=14), EAAC1 −/− (n=15), **p=2.1e-3). Data in panels ?,?,? represent the band intensity ratio between the target protein and β-actin in samples from WT versus EAAC1 7 ' mice in the same blotting membrane.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Western blots for D1R, D2R and β-actin, in the presence of the mGluRI blockers LY367385 (50 μM) and MPEP (10 μM), show no significant difference in the expression of D1R and D2R (D1R: WT (n=9), EAAC1 −/− (n=9), p=0.35; D2R: WT (n=7), EAAC1 −/− (n=7), p=0.064). (B) mGluRI blockade induces a slight reduction in DARPP-32 expression between WT (n=9) and EAAC1 −/− mice (n=9, *p=0.029). (C) Western blot analysis for pDARPP-32 T34 (WT (n=5), EAAC1 −/− (n=6), p=0.98), pDARPP-32 T75 (WT (n=6), EAAC1 −/− (n=7), *p=0.017), pDARPP-32 S97 (WT (n=9), EAAC1 −/− (n=9), p=0.34), pDARPP-32 S130 (WT (n=12), EAAC1 −/− (n=9), **p=4.6e-3). (D) Pie chart representation of the phosphorylation distribution on the T34, T75, S97 and S130 sites of DARPP-32 in the presence of mGluRI blockers. The red curves highlight the T75 and S130 site, which show reduced phosphorylation in EAAC1 −/− mice. (E) As in (C), following data normalization by the average band intensity values measured in WT mice (WT (n=5), EAAC1 −/− (n=6), p=0.62), pDARPP-32 T75 (WT (n=9), EAAC1 −/− (n=7), ***p=1.0e-5), pDARPP-32 S97 (WT (n=9), EAAC1 −/− (n=9), p=0.16), pDARPP-32 S130 (WT (n=12), EAAC1 −/− (n=9), ***p=2.1e-4). Data in panels A,B,E represent the band intensity ratio between the target protein and β-actin measured in samples from EAAC1 −/− mice and normalized by analogous measures in samples from WT mice blotted in the same membrane.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Neuronal glutamate transporters control dopaminergic signaling and compulsive behaviors

doi: 10.1101/224477

Figure Lengend Snippet: (A) Timeline of the experimental design. At P14-16, mice received a unilateral stereotaxic injection of hM3D(Gq). After one week, they started receiving daily I/P saline injections. At P28-30, they received I/P injections of CNO (5 mg/Kg). One hour after the CNO injections, they were video-monitored to examine their grooming behavior. Two hours after the CNO injections, they were sacrificed. Proteins for Western blot analysis were extracted from the control and injected striatum and from the adjacent cortices. (B) Left: mCherry expression in D1 Cre/+ mice. A significant increase in mCherry expression was detected only in the striatum of D1 Cre/+ mice (n=10, *p=0.040). Right: mCherry expression in A2A Cre/+ mice. A significant increase in mCherry expression was detected only in the striatum of A2A Cre/+ mice (n=23, *p=0.035). (C) Left: D1R and D2R expression in D1 Cre/+ mice (D1R: n=9, *p=0.013; D2R: n=8, p=0.35). The expression of D1R is significantly reduced in the injected striatum. Right: D1R and D2R expression in A2A Cre/+ mice (D1R: n=9, p=0.63; D2R: n=5, p=0.34). The expression of D1R and D2R is similar in the injected and non-injected striatum. (D) Left: hM3D(Gq) injection in D1 Cre/+ mice leads to increased pDARPP-32 S130 (pDARPP-32 T34 n=10, p=0.92; pDARPP-32 T75 n=11, p=0.18; pDARPP-32 S97 n=11, p=0.13, pDARPP-32 S130 n=11, **p=5.6e-3). Right: hM3D(Gq) injection in A2A Cre/+ mice leads to no change in pDARPP-32 (pDARPP-32 T34 n=9, p=0.75; pDARPP-32 T75 n=8, p=0.84; pDARPP-32 S97 n=8, p=0.31, pDARPP-32 S130 n=6, p=0.89). (E) Summary of the frequency (Frequency: Sham (n=25), D1 Cre/+ (n=39), Sham vs D1 Cre/+ **p=6.0e-3, A2A Cre/+ (n=49), Sham vs A2A Cre/+ p=0.28) and duration of grooming episodes in Sham and D1 Cre/+ mice injected with hM3D(Gq) (Duration: Sham (n=34), D1 Cre/+ (n=44), Sham vs D1 Cre/+ p=0.12, A2A Cre/+ (n=58), Sham vs A2A Cre/+ p=0.08). (F) Relationship between the frequency and duration of grooming episodes in Sham (left), D1 Cre/+ (middle) and A2A Cre/+ mice (right). The inset represents a density plot of the data, with blue areas corresponding to the duration and frequency of the most commonly observed grooming episodes.

Article Snippet: We used the following primary antibodies: rabbit anti D1R and D2R (1:200, Cat# ADR-001 and ADR-002 respectively; Alomone Labs, Jerusalem, Israel); rabbit anti mGluR5/1a (1:500, Cat# 2032-mGluR5/1a; PhosphoSolutions, Aurora, CO); rabbit anti GLAST (1:1,000, Cat# 5684; Cell Signaling Technology, Danvers, MA); rabbit anti GLT-1 (1:1,000, Cat# 3838; Cell Signaling Technology, Danvers, MA); rabbit anti DARPP-32 (1:1,000, Cat# 2306; Cell Signaling Technology, Danvers, MA); rabbit antibodies against different pDARPP-32 isoforms (pDARPP-32 T34 , 1:500, Cat# 12438; Cell Signaling Technology, Danvers, MA), (pDARPP-32 T75 and pDARPP-32 S97 , 1:1,000, Cat# 2301 and Cat# 3401 respectively; Cell Signaling Technology, Danvers, MA), (pDARPP-32 S130 , 1:500, Cat# p1025-137; PhosphoSolutions, Aurora, CO), mCherry (Cat# 600-401-P16 Rockland Antibodies & Assays, Limerick, PA) and β-actin (1:1,000, Cat# 4970, Cell Signaling Technology, Danvers, MA).

Techniques: Injection, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Gestational stress disrupts dopamine and oxytocin signaling in the postpartum reward system of rats: implications for mood, motivation and mothering

doi: 10.1038/s41598-024-84043-6

Figure Lengend Snippet: Experimental timeline. Pregnant rats were exposed to a chronic variable stress procedure from gestation day 7 (GD7)-GD20. No stress controls were handled daily. The day of birth (DOB) was designated as postpartum day 0 (PD0). Behavioral testing and brain collection occurred from PD2-PD10. In Expt. 1 , rats (No Stress = 10; Stress = 9) underwent testing for maternal behavior and brains were collected on PD7 for qPCR gene expression analysis of oxytocin (OT) and oxytocin receptor (OTR) in the hypothalamus and ventral tegmental area (VTA). In Expt. 2, rats (No Stress = 6; Stress = 5) were tested on the conditioned place preference (CPP) paradigm. In Expt. 3 , rats (No Stress = 9; Stress = 9) underwent testing on the sucrose preference test (SPT) and brains were collected on PD8 for immunohistochemistry (IHC) and densiometric analysis of dopamine (DA) markers (DAT, dopamine transporter; D1R; dopamine 1 receptor; D2R, dopamine 2 receptor; TH, tyrosine hydroxylase) in the NAc. Maternal and litter characteristics were measured in the SPT dams. In Expt. 4 , rats (No Stress = 12; Stress = 10) were tested on the elevated plus maze (EPM) and forced swim test (FST) and brains were collected on PD10 for IHC and densiometric analysis of additional DA markers in the NAc (pTH, phosphorylated tyrosine hydroxylase; VMAT, vesicular monoamine transporter) as well as counts of TH + cells in the VTA. OT IHC was also done to assess the effects of gestational stress on OT + cells in the hypothalamus and OT fibers in the VTA. In Expt. 5 , dams (No Stress = 6; Stress = 6), the NAc was dissected on PD6 for Liquid Chromatography-Mass Tandem Spectrometry (LC–MS/MS) to measure DA content and the dopamine metabolite, DOPAC. Figure created in https://BioRender.com .

Article Snippet: Next, sections were blocked in 5% BSA in 0.1 M PBS for 1 h at RT and incubated with one of the following primary antibodies: rabbit anti-D1R (1:50; Cat#sc-14001, Santa Cruz Biotechnology), goat anti-dopamine D2R (1:100; Cat#sc-7522, Santa Cruz Biotechnology), rabbit anti-TH (1:1000; Cat#AB152, Millipore), rabbit anti-DAT (1:200; Cat#AB1591P, Millipore), rabbit anti-VMAT(1:100; Cat#PA5-22,864, ThermoFisher), and rabbit anti- pTH (1:500; Cat#AB5935, Millipore) overnight at 4 °C.

Techniques: Gene Expression, Conditioned Place Preference, Immunohistochemistry, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific Reports

Article Title: Gestational stress disrupts dopamine and oxytocin signaling in the postpartum reward system of rats: implications for mood, motivation and mothering

doi: 10.1038/s41598-024-84043-6

Figure Lengend Snippet: Gestational stress alters markers of dopaminergic signaling in the NAc shell. ( A ) In the nucleus accumbens (NAc) shell, markers associated with dopaminergic signaling were examined via immunohistochemistry on PD8 or PD10. Compared to unstressed controls, dams exposed to gestational stress showed a reduction in percent area staining for several dopaminergic markers including ( B ) tyrosine hydroxylase, TH, ( C ) phosphorylated TH, pTH and ( D ) the dopamine transporter, DAT. ( E ) There was no effect of gestational stress on vesicular monoamine transporter, VMAT. Percent area staining for dopamine receptors ( F ) D1R and ( G ) D2R in the NAc shell was also examined and only the D2R was reduced by gestational stress. Representative images of each marker analyzed are shown in ( A ). * p < 0.05; ** p < 0.005; *** p ≤ 0.0005.

Article Snippet: Next, sections were blocked in 5% BSA in 0.1 M PBS for 1 h at RT and incubated with one of the following primary antibodies: rabbit anti-D1R (1:50; Cat#sc-14001, Santa Cruz Biotechnology), goat anti-dopamine D2R (1:100; Cat#sc-7522, Santa Cruz Biotechnology), rabbit anti-TH (1:1000; Cat#AB152, Millipore), rabbit anti-DAT (1:200; Cat#AB1591P, Millipore), rabbit anti-VMAT(1:100; Cat#PA5-22,864, ThermoFisher), and rabbit anti- pTH (1:500; Cat#AB5935, Millipore) overnight at 4 °C.

Techniques: Immunohistochemistry, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Colony-Stimulating Factor 1 Receptor by PLX3397 Prevents Amyloid Beta Pathology and Rescues Dopaminergic Signaling in Aging 5xFAD Mice

doi: 10.3390/ijms21155553

Figure Lengend Snippet: Schematic diagram illustrating the experimental groups and protocol. When the 5xFAD mice were 9 months old, they were administered PLX3397 (50 mg·kg −1 , p.o. ) or vehicle for 30 days. Then, each group underwent positron emission tomography (PET) imaging targeting the dopamine D2 receptor (D2R) and metabotropic glutamate receptor 5 (mGluR5), and histopathological and molecular analyses were also performed.

Article Snippet: The washed tissues were then incubated with the following primary antibodies overnight at 4 °C: Anti-rabbit D2R (cat no. AB5084P; 1:500 dilution; Merck, Darmstadt, Germany), anti-rabbit TH (cat no. NB300-109; 1:1000 dilution; Novus, Centennial, CO, USA), or anti-rabbit DAT (cat no. D6944; 1:000 dilution; Sigma-Aldrich) antibodies.

Techniques: Positron Emission Tomography, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Colony-Stimulating Factor 1 Receptor by PLX3397 Prevents Amyloid Beta Pathology and Rescues Dopaminergic Signaling in Aging 5xFAD Mice

doi: 10.3390/ijms21155553

Figure Lengend Snippet: Neuro-PET imaging for evaluating dopamine D2 receptor (D2R) and metabotropic glutamate receptor 5 (mGluR5) expression following PLX3397 treatment. ( A ) Representative nondisplaceable binding potential (BPND) parametric images for WT and Tg mice. Regional brain BPND levels in Tg mice after PLX3397 administration are shown. ( B ) Quantification of BPND for mGluR5 and D2R in the WT, Tg, and Tg + PLX3397 groups. * p < 0.05 WT vs. Tg; # p < 0.05 Tg vs. Tg + PLX3397 group. Error bars indicate SD ( n = 4/group).

Article Snippet: The washed tissues were then incubated with the following primary antibodies overnight at 4 °C: Anti-rabbit D2R (cat no. AB5084P; 1:500 dilution; Merck, Darmstadt, Germany), anti-rabbit TH (cat no. NB300-109; 1:1000 dilution; Novus, Centennial, CO, USA), or anti-rabbit DAT (cat no. D6944; 1:000 dilution; Sigma-Aldrich) antibodies.

Techniques: Imaging, Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Colony-Stimulating Factor 1 Receptor by PLX3397 Prevents Amyloid Beta Pathology and Rescues Dopaminergic Signaling in Aging 5xFAD Mice

doi: 10.3390/ijms21155553

Figure Lengend Snippet: Effects of PLX3397 on the dopaminergic pathway. Representative immunohistochemical images for WT, Tg, and Tg + PLX3397 mice showing staining for dopamine D2 receptor (D2R) ( A ), tyrosine hydroxylase (TH) ( B ), and dopamine transporter (DAT) ( C ) and the quantification of D2R, TH, and DAT levels in the striatum region of the mouse brain ( n = 4 for TG group, n = 5 for WT and Tg + PLX3397 group). * p < 0.05 WT vs. Tg; # p < 0.05, ## p < 0.01 Tg vs. Tg + PLX3397 group. Error bars indicate SD.

Article Snippet: The washed tissues were then incubated with the following primary antibodies overnight at 4 °C: Anti-rabbit D2R (cat no. AB5084P; 1:500 dilution; Merck, Darmstadt, Germany), anti-rabbit TH (cat no. NB300-109; 1:1000 dilution; Novus, Centennial, CO, USA), or anti-rabbit DAT (cat no. D6944; 1:000 dilution; Sigma-Aldrich) antibodies.

Techniques: Immunohistochemical staining, Staining

Journal: Brain Sciences

Article Title: Transient Chemogenetic Inhibition of D1-MSNs in the Dorsal Striatum Enhances Methamphetamine Self-Administration

doi: 10.3390/brainsci9110330

Figure Lengend Snippet: ( a ) Schematic representation of a coronal section through the dorsal striatum of the adult rat brain indicating the placement of injector needle for virus infusions. ( b ) Schematic of the lentiviral vector backbone indicating the genes of interest along with the dynorphin (Dyn) promoter that are inserted upstream of the WPRE in the pHIV-7 vector; IRES, internal ribosome entry site; eGFP, enhanced green fluorescent protein. ( c , d ) Time course of Dyn-GFP virus infection demonstrated that maximal expression was seen between 3–6 weeks after virus injection. ( d ) Quantitative analysis of Dyn-GFP positive cells in the striatum of virus injected animals; n = 2–4 each time point. Data is represented as mean ± SEM. ( e – j ) Coronal sections with Dyn-GFP positive neurons along the rostral-caudal direction of the dorsal striatum. The rectangular box in f, h indicates the striatal area labeled with Dyn-GFP neurons in g, i. LV, lateral ventricle; DS, dorsal striatum; cc, corpus callosum. ( k – m ) Colabeling of Dyn-GFP with SubP (CY3, red) a maker for D1R-MSNs; arrow and arrowheads in k–m point to colabeled immunoreactive cells. ( n – p ) Colabeling of Dyn-GFP with ENK (CY3, red) a maker for D2R-MSNs; arrowhead in n–p point to Dyn-GFP cell that is not colabeled with ENK cells. ( q – s ) Confocal z-stack images in orthogonal view indicating colabeling of the cell in ( k – m ) pointed with an arrow. Xy- and yz axis is indicated in q-s to demonstrate equal penetration of GFP and SubP antibodies. ( t , u ) Confocal images indicating detector gain ( t ; black and white image shows no red—overmodulation or green—undermodulation of cells and therefore the lasers have been optimized in the multi-channel image acquisition) and amplifier gain ( u ; rainbow image shows no red—overmodulation or blue—undermodulation of areas expressing cells; note that the area of the axon bundles are blue due to lack of any cellular bodies) of the section used for orthogonal view. GFP, green fluorescent protein; SubP, substance P; ENK, enkephalin. ( v ) Virus injected section stained with Vector FastRed showing minimal damage to the dorsal striatum. ( w , x ) Iba-1 staining via DAB in virus naïve ( w ) and Dyn-GFP ( x ) injected rat. ( y , z ) GFP immunoreactivity in the dorsal striatum of a rat injected with Dyn-hM4D-GFP ( y ) and Dyn-rM3D-GFP ( z ). Scale bar in u is 200 um applies to e, g, i, j, v, w, x; 20 um applies k–p; 30 um applies q–u; 70 um applies y–z.

Article Snippet: Blots were blocked with 2.5% bovine serum albumin (for phosphoproteins) or 5% milk (w/v) in TBST (25 mM Tris–HCl (pH 7.4), 150 mM NaCl and 0.1% Tween 20 (v/v)) for 16–20 h at 4 °C and were incubated with the primary antibody for 16–20 h at 4 °C: antibody to phosphorylated-p44/42 MAPK (pErk1/2) at Thr202/Tyr204 (mouse monoclonal, 1:1000, Cell Signaling cat# 9106S, molecular weights 44/42 kDa); total Erk1/2 (rabbit polyclonal, 1:4000, Cell Signaling cat# 9102, molecular weights 44/42 kDa); pCamKII Tyr-286 (rabbit polyclonal, 1:1000, Abcam cat# ab5683, molecular weight 50 kDa); total CaMKII (rabbit polyclonal, 1:2000, Abcam cat# ab52476, molecular weight 47 kDa); pAkt Ser-473 (rabbit polyclonal, 1:1000, Cell Signaling cat# 4060S, molecular weight 60 kDa); total Akt (rabbit polyclonal, 1:1000, Cell Signaling cat# 4691S, molecular weight 60 kDa); D1R (rabbit polyclonal, 1:1000, Abcam cat# ab20066, molecular weight 48 kDa); Cav-1 (rabbit monoclonal, 1:1000, Cell Signaling cat# 3267S, molecular weight 24 kDa); PSD-95 (rabbit polyclonal, 1:500, Millipore cat# 04-1066, molecular weight 95 kDa); Dopamine D2 receptors (D2R, rabbit polyclonal, 1:1000, Sigma Aldrich cat# AB5084P, molecular weight 48 kDa); dopamine transporter (DAT; rat monoclonal, 1:500, Santacruz cat# sc-32258, molecular weight 90 kDa).

Techniques: Plasmid Preparation, Infection, Expressing, Injection, Labeling, Staining