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: 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, Analysis, Expressing, Phospho-proteomics, Membrane

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, Analysis, Phospho-proteomics, Membrane

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, Saline, Western Blot, Analysis, Control, Expressing, FACS

Journal: Frontiers in Cellular Neuroscience

Article Title: Inhibitory Effects of Trifluoperazine on Peripheral Proinflammatory Cytokine Expression and Hypothalamic Microglia Activation in Obese Mice Induced by Chronic Feeding With High-Fat-Diet

doi: 10.3389/fncel.2021.752771

Figure Lengend Snippet: List of reagents (antibodies and assay kits) in the study.

Article Snippet: Polyclonal rabbit anti-D2R , Biorbyt, Cat# orb10515, RRID:AB_10747533.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in molecular neuroscience

Article Title: Repeated inhibition of sigma-1 receptor suppresses GABA A receptor expression and long-term depression in the nucleus accumbens leading to depressive-like behaviors.

doi: 10.3389/fnmol.2022.959224

Figure Lengend Snippet: FIGURE 1 Repeated injection of NE-100 in the NAc induces depressive-like behaviors. (A) The time course of the experimental procedure. (B) Schematic diagram of the NAc (left panel) and image of the NAc injected with Evans-blue (right panel). (C) The level of σ1R mRNA in the NAc was examined at the end of the behavioral tests (n = 8 mice per group; Student’s t-test). (D) Representative Western blots of biotin-labeled surface proteins (surface) and total proteins (total) σ1R in the NAc. Na+-K+ ATPase served as internal control and GAPDH served as a negative control. Bar graphs indicate the ratio of proteins at the cell surface to their total levels. **p < 0.01 vs. control mice (n = 8 mice per group; Student’s t-test). (E) Bar graphs show the distance traveled in OFT in control mice and NE-100 mice (n = 12 mice per group; Student’s t-test). (F,G) Bar graphs show the immobility time of FST and TST in control mice and NE-100 mice. **p < 0.01 vs. control mice (n = 11 mice in control group of TST and n = 12 mice in other groups; Student’s t-test).

Article Snippet: Several antibodies were used in this study: anti-D2R (1:1,000; Millipore Cat#AB5084p, RRID:AB_2094980), anti-GABAAR-α1 (Sigma Cat#G4416, RRID:AB_477016), anti-GABAARα2(AbcamCat#ab72445, RRID:AB_1268929), anti-GABAAR-β2 (Affinity Biosciences Cat#DF6671, RRID:AB_2838633), antiGABAAR-β3 (Abcam Cat#ab98968, RRID:AB_10670809), anti-phosphorylation PKC (Epsilon Ser729; Abcam Cat#ab63387, RRID:AB_1142277), anti-PKC (Abcam Cat#ab253274, RRID:AB_2827663), anti-σ1R (B5; Santa Cruz Biotechnology Cat#sc-137075, RRID:AB_2285870), anti-Na+-K+ ATPase (Cell Signaling Technology Cat#3010, RRID:AB_2060983) and anti-GAPDH (Abcam Cat#ab181602, RRID:AB_2630358).

Techniques: Injection, Western Blot, Labeling, Control, Negative Control

Journal: Frontiers in molecular neuroscience

Article Title: Repeated inhibition of sigma-1 receptor suppresses GABA A receptor expression and long-term depression in the nucleus accumbens leading to depressive-like behaviors.

doi: 10.3389/fnmol.2022.959224

Figure Lengend Snippet: FIGURE 3 Repeated injection of NE-100 suppresses GABAAR and D2R expression. (A) The levels of GABAAR-α1-5, -β1-3, -γ1-3, -δ subunits, and D1R, D2R mRNA in the NAc were examined at the end of the behavioral tests. *p < 0.05 vs. control mice (n = 8 mice per group; two-way ANOVA). (B) Bar graphs show levels of GABAAR-α1, GABAAR-α2, GABAAR-β2, and GABAAR-β3 protein in the NAc normalized by the level of GAPDH were normalized by control levels. *p < 0.05 vs. control mice (n = 8 mice per group; two-way ANOVA). (C) Representative Western blots of biotin- labeled surface proteins (surface) and total proteins (total) D2R in the NAc. Na+-K+ ATPase served as internal control and GAPDH served as a negative control. Bar graphs indicate the ratio of proteins at the cell surface to their total levels. **p < 0.01 vs. control mice (n = 8 mice per group; Student’s t -test).

Article Snippet: Several antibodies were used in this study: anti-D2R (1:1,000; Millipore Cat#AB5084p, RRID:AB_2094980), anti-GABAAR-α1 (Sigma Cat#G4416, RRID:AB_477016), anti-GABAARα2(AbcamCat#ab72445, RRID:AB_1268929), anti-GABAAR-β2 (Affinity Biosciences Cat#DF6671, RRID:AB_2838633), antiGABAAR-β3 (Abcam Cat#ab98968, RRID:AB_10670809), anti-phosphorylation PKC (Epsilon Ser729; Abcam Cat#ab63387, RRID:AB_1142277), anti-PKC (Abcam Cat#ab253274, RRID:AB_2827663), anti-σ1R (B5; Santa Cruz Biotechnology Cat#sc-137075, RRID:AB_2285870), anti-Na+-K+ ATPase (Cell Signaling Technology Cat#3010, RRID:AB_2060983) and anti-GAPDH (Abcam Cat#ab181602, RRID:AB_2630358).

Techniques: Injection, Expressing, Control, Western Blot, Labeling, Negative Control