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: Theranostics

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

doi: 10.7150/thno.83079

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

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

Techniques: Activity Assay, Expressing, Saline

Journal: Theranostics

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

doi: 10.7150/thno.83079

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

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

Techniques: Activation Assay, Injection

Journal: Theranostics

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

doi: 10.7150/thno.83079

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

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

Techniques: Activation Assay, Virus, Control, Transfection

Journal: Theranostics

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

doi: 10.7150/thno.83079

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

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

Techniques: Activation Assay, Expressing

Journal: Theranostics

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

doi: 10.7150/thno.83079

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

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

Techniques: Activation Assay, Blocking Assay, Virus

Journal: Brain, behavior, and immunity

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

doi: 10.1016/j.bbi.2017.05.004

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

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

Techniques: Immunohistochemistry, Labeling