mouse monoclonal anti a2ar  (Santa Cruz Biotechnology)

Journal: Current protocols in neuroscience

Article Title: Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay

doi: 10.1002/cpns.86

Figure Lengend Snippet: Diagram of PLA_BF with secondary antibody-conjugated PLA probes. Single PLA was performed with one primary antibody (i.e. rabbit anti-D2R or mouse anti-A2AR) and a pair of PLA probes to the primary antibody (A). Dual PLA was performed with two primary antibodies from two different species and the species specific PLA probes (B). For a discussion of the impact of using polyclonal antibodies on the possible binding configuration, see the main text (Introduction and Understanding the PLA results).

Article Snippet: Sections from formalin-fixed, paraffin-embedded human brain tissue mounted on glass slides Phosphate buffer (PB) pH 7.4 Xylene * 100%, 95%, 80%, 75% and 50% ethanol (anhydrous denatured, histological grade) Deionized water (DI H 2 O) Tris buffer with sodium (TBS), pH 7.4 Tris buffer with sodium and tween-20 (TBS-T), pH 7.4 10% Tween-20 Antigen retrieval buffer: 10 mM Sodium Citrate, pH 6, with 0.02% Tween-20 Quenching buffer: 1 % hydrogen peroxide (H 2 O 2 ) in TBS (Store hydrogen peroxide in the dark at 4 °C) Antibodies: Dopamine receptor D2R was probed using a rabbit polyclonal anti-D2R (Millipore Cat# ABN462, RRID: AB_2810225) and adenosine receptor A2AR was detected using a mouse monoclonal anti-A2AR antibody (Millipore 05717, RRID: AB_11213750).

Techniques: Binding Assay

Journal: Current protocols in neuroscience

Article Title: Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay

doi: 10.1002/cpns.86

Figure Lengend Snippet: Representative images of single PLA, dual PLA, and negative controls from human brain sections. Expression of D2R, A2AR (by single PLA) and A2AR-D2R (by dual PLA) was detected in the striatum (A-C). None or very few PLA puncta were detected in the negative controls that omitted the primary antibody in the single PLA (D-E) or anti-A2AR in the dual PLA (F). Scale bar, 25 μm. The arrows indicated representative PLA puncta. The images were obtained with an Olympus BX51 and a QImaging 1600X1200 pixel 8-bit color CCD camera controlled by Stereo Investigator (MBF Bioscience).

Article Snippet: Sections from formalin-fixed, paraffin-embedded human brain tissue mounted on glass slides Phosphate buffer (PB) pH 7.4 Xylene * 100%, 95%, 80%, 75% and 50% ethanol (anhydrous denatured, histological grade) Deionized water (DI H 2 O) Tris buffer with sodium (TBS), pH 7.4 Tris buffer with sodium and tween-20 (TBS-T), pH 7.4 10% Tween-20 Antigen retrieval buffer: 10 mM Sodium Citrate, pH 6, with 0.02% Tween-20 Quenching buffer: 1 % hydrogen peroxide (H 2 O 2 ) in TBS (Store hydrogen peroxide in the dark at 4 °C) Antibodies: Dopamine receptor D2R was probed using a rabbit polyclonal anti-D2R (Millipore Cat# ABN462, RRID: AB_2810225) and adenosine receptor A2AR was detected using a mouse monoclonal anti-A2AR antibody (Millipore 05717, RRID: AB_11213750).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but Not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration

doi: 10.3390/ijms20236100

Figure Lengend Snippet: ( A ) A molecular model is shown of the A2AR-D2R heterodimer to illustrate the that the A2AR synthTM2 is not part of the interface (The PDB coordinate of this molecular model was obtained from Borroto-Escuela et al. ). ( B , C ) Effects of intra-accumbal microinjections of the rat A2AR synthTM2 peptide or vehicle in the presence of CGS 21680 during cocaine self-administration on the density of the PLA positive A2AR-D2R heteroreceptor complexes in nucleus accumbens core (AcbC), nucleus accumbens shell (AcbSh,) and caudate putamen (CPU). ( B ) Representative examples are given of the densities of the red PLA positive A2AR-D2R heteroreceptor complexes after the rat A2AR synthTM2 peptide treatment vs. vehicle in the ventral striatum. ( C ) The microinjection of the A2AR synthTM2 peptide is shown to not alter the density of the PLA positive complexes per nucleic per cell in any of the regions analyzed. Bregma 1.00 mm, Scale bar is 30 μm. Mean ± SEM, number of rats = 5 per group. Student’s t -test with Bonferoni correction.

Article Snippet: Free-floating formalin fixed brain sections (30 μm-thick, cut using a cryostat) at Bregma level (1.0 mm) from rats after cocaine-self administration were employed using the following primary antibodies: rabbit monoclonal anti-A2AR (AB1559F, 1:250; Millipore, Sweden), mouse monoclonal anti-A2AR (05-717, 1:500, Millipore, Sweden), and the mouse monoclonal anti-D2R (MABN53, 1:600, Millipore, Sweden).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but Not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration

doi: 10.3390/ijms20236100

Figure Lengend Snippet: Effects of intra-accumbal microinjections of the rat A2AR synthTM2 peptide or vehicle in the presence of CGS 21680 during cocaine self-administration on the density of the PLA positive A2AR-A2AR homoreceptor complexes in nucleus accumbens core (AcbC), nucleus accumbens shell (AcbSh), and caudate putamen (CPU). ( A ) Representative examples is given for the reduction of the densities of the red PLA positive A2AR-A2AR homoreceptor complexes after the rat A2AR synthTM2 peptide treatment vs. vehicle in the ventral striatum. ( B ) The microinjection of the A2AR synthTM2 peptide is shown to produce a significant reduction in the density of the PLA positive complexes per nucleic per cell in the AcbC and AcbSh. Bregma 1.00 mm, Scale bar is 30 μm. Mean ± SEM, number of rats = 5 per group. Student’s t -test with Bonferoni correction.

Article Snippet: Free-floating formalin fixed brain sections (30 μm-thick, cut using a cryostat) at Bregma level (1.0 mm) from rats after cocaine-self administration were employed using the following primary antibodies: rabbit monoclonal anti-A2AR (AB1559F, 1:250; Millipore, Sweden), mouse monoclonal anti-A2AR (05-717, 1:500, Millipore, Sweden), and the mouse monoclonal anti-D2R (MABN53, 1:600, Millipore, Sweden).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but Not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration

doi: 10.3390/ijms20236100

Figure Lengend Snippet: [ 3 H]-raclopride/quinpirole competition experiments to determine changes in D2R affinities induced by adenosine A2AR agonist CGS-21680 in the saline-injected (vehicle) rat group (control group). ( A ) Competition experiments involving dopamine D2-likeR antagonist [ 3 H]-raclopride binding versus increasing concentrations of quinpirole were performed in ventral striatal membrane preparations from the control group (60 µg/mL) in the presence or absence of the adenosine A2AR agonist CGS 21680 (100 nM) as indicated. Nonspecific binding was defined as the binding in the presence of 100 μM (+)-butaclamol. [ 3 H]-raclopride/quinpirole competition curve is based on the values of four rats with each experiment performed in triplicates. The binding values are given in percent of specific binding at the lowest concentration of quinpirole employed. ( B ) Analysis and presentation are given of the A2AR agonist CGS 21680 (100 nM) induced changes in the high-affinity value (K i , High ), low-affinity value (K i , Low ), and proportion of receptors in the high-affinity state (RH). Means ± SEM are given from four independent experiments performed in triplicates. Statistical analysis was performed by paired Student’s t-test (* p < 0.015): the group of rats treated with CGS 21680 is significantly different compared to the group receiving only the saline solution. No significant differences in low-affinity values were observed after CGS-21680 modulation compared to vehicle ( p = 0.0571 by paired t -test). Furthermore, no significant differences in proportion of receptors in the high-affinity state were observed after CGS-21680 modulation compared to vehicle ( p = 0.5760 paired t -test).

Article Snippet: Free-floating formalin fixed brain sections (30 μm-thick, cut using a cryostat) at Bregma level (1.0 mm) from rats after cocaine-self administration were employed using the following primary antibodies: rabbit monoclonal anti-A2AR (AB1559F, 1:250; Millipore, Sweden), mouse monoclonal anti-A2AR (05-717, 1:500, Millipore, Sweden), and the mouse monoclonal anti-D2R (MABN53, 1:600, Millipore, Sweden).

Techniques: Injection, Binding Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but Not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration

doi: 10.3390/ijms20236100

Figure Lengend Snippet: [ 3 H]-raclopride/quinpirole competition experiments to determine changes in D2R affinities induced by adenosine A2AR agonist CGS-21680 in the TM2 rat group. ( A ) Competition experiments involving dopamine D2-likeR antagonist [ 3 H]-raclopride binding versus increasing concentrations of quinpirole were performed in ventral striatal membrane preparations from the TM2 injected rat group (60 µg/mL) in the presence or absence of the adenosine A2AR agonist CGS-21680 (100 nM) as indicated. Nonspecific binding was defined as the binding in the presence of 100 μM (+)-butaclamol. [ 3 H]-raclopride/quinpirole competition curve is based on the values of four rats with each experiment performed in triplicates. The binding values are given in percent of specific binding at the lowest concentration of quinpirole employed. ( B ) Analysis and presentation are given of the A2AR agonist CGS-21680 (100 nM) induced changes in the high-affinity value (K i , High ), low-affinity value (K i , Low ), and proportion of receptors in the high-affinity state (RH). Means ± SEM are given from four independent experiments performed in triplicates. Statistical analysis was performed by paired Student’s t -test (* p < 0.019): the group of rats treated with CGS-21680 is significantly different compared to the group receiving only the TM2. No significant differences in low-affinity values were observed in rats receiving TM2 + CGS-21680 compared to rats receiving only TM2 ( p = 0.0506 by paired t -test). Also, no significant differences in proportion of receptors in the high-affinity state were observed in rats receiving TM2 + CGS-21680 compared to rats receiving only TM2 ( p = 0.0514 paired t -test).

Article Snippet: Free-floating formalin fixed brain sections (30 μm-thick, cut using a cryostat) at Bregma level (1.0 mm) from rats after cocaine-self administration were employed using the following primary antibodies: rabbit monoclonal anti-A2AR (AB1559F, 1:250; Millipore, Sweden), mouse monoclonal anti-A2AR (05-717, 1:500, Millipore, Sweden), and the mouse monoclonal anti-D2R (MABN53, 1:600, Millipore, Sweden).

Techniques: Binding Assay, Injection, Concentration Assay

Journal: PLoS ONE

Article Title: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

doi: 10.1371/journal.pone.0199601

Figure Lengend Snippet: A &B) A2AR +/+ enhanced the Th17 response, but A2AR -/- γδ T cells did not; and neither enhanced the Th1 response. A2AR +/+ and A2AR -/- γδ T cells were isolated from immunized wt-B6 or A2AR -/- mice, respectively. Responder αβ T cells (1 x 10 6 /well) prepared from IRBP 1-20 immunized TCR-δ -/- mice on day 13 post-immunization were stimulated for 5 days with immunizing antigen and APCs under Th17 (A) of Th1 (B) polarized conditions with or without addition of 2% (2 x 10 /well) A2AR +/+ or A2AR -/- γδ T cells. The numbers of αβTCR + IL-17 + and αβTCR + IFN-γ + cells were calculated by FACS analysis after a 5-day in vitro stimulation. C) Addition of A2AR +/+ γδ T cells, but not of A2AR -/- γδ T cells, to αβ responder T cells enhanced IL-17 production but did not affect IFN-γ production. Production of IL-17 and IFN-γ by immunized TCR-δ -/- mice was assessed by ELISA, after in vitro stimulation with the immunizing peptide (IRBP 1-20 ), in the presence of A2AR +/+ or A2AR -/- γδ T cells (2%). Cytokine amounts were evaluated in the culture cell supernatants by ELISA after a 48 h stimulation. D) Treatment of A2AR antagonist abolished the enhancing effect of A2AR +/+ γδ T cells. A2AR +/+ γδ T cells were added to responder αβ T cells in culture, with or without a pre-incubation with SCH58261, a selective A2AR antagonist (100nM) [ , ]. Results show that blockade of A2AR ligation abolished the enhancing effect of A2AR +/+ γδ T cells. E) Administration of TCR-δ -/- mice with A2AR +/+ γδ T cells, but not with A2AR -/- γδ T cells, enhanced the Th17 response. Groups (n = 4) of TCR-δ -/- mice were injected with A2AR +/+ or A2AR -/- (1 x 10 6 /mouse) γδ T cells, before they were immunized with a pathogenic dose of IRBP 1-20 /CFA. At 13 days post-immunization, the Th17 responses were assessed 5 days after in vitro stimulation. The results shown are representative of those from five experiments. Differences were considered significant when P ≤ 0.05 (asterisk) and very significant when P ≤ 0.01 (two asterisks). F.) TCR-δ -/- mice injected with A2AR +/+ , but not A2AR -/- , γδ T cells were more susceptible to induced EAU. Groups of TCR-δ -/- mice were immunized with IRBP 1-20 /CFA with or without a prior injection of 1 x 10 5 (per mouse) A2AR +/+ or A2AR -/- γδ T cells. EAU was scored as previously reported [ , , ]. The results of two separate experiments are shown.

Article Snippet: PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, In Vitro, Enzyme-linked Immunosorbent Assay, Incubation, Ligation, Injection

Journal: PLoS ONE

Article Title: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

doi: 10.1371/journal.pone.0199601

Figure Lengend Snippet: A) A2AR agonist inhibited TCR-δ -/- responder T cells more effectively than it inhibited the B6 responder T cells. The αβ responder T cells (1 x 10 6 /well) were isolated from IRBP 1-20 -immunized B6 (upper panels) or TCR-δ -/- mice (lower panels). The CFSE-labeled αβ T cells were stimulated for 5 d with immunizing peptide and APCs in the absence or presence of A2AR agonist. Cell proliferation was assessed by FACS analysis. B) In vitro treatment of A2AR agonist enhanced the Th17 responses when added to B6 responder T cells but inhibited the Th17 responses when added to TCR-δ -/- responder T cells. Comparison of IL-17 and IFN-γ production by immunized αβ T cells isolated from immunized B6 or TCR-δ -/- mice after in vitro stimulation, in the absence or presence of A2AR agonist, showing that A2AR agonist inhibited the Th1 response but enhanced the Th17 response of B6 cells, but not of TCR-δ -/- T cells.

Article Snippet: PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, Labeling, In Vitro, Comparison

Journal: PLoS ONE

Article Title: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

doi: 10.1371/journal.pone.0199601

Figure Lengend Snippet: A) Activated γδ T cells express an increase of >1000-times the A2AR but only minimally increased A2BR. Activated γδ T cells and naïve γδ T cells were isolated from immunized from naïve B6 mice, respectively. The expression of A2AR/A2BR was determined by real-time PCR assay. Real-time RT-PCR analysis of A2AR and A2B transcripts among total RNA isolated from αβ and γδ T cells isolated from naïve and IRBP 1-20 -immunized B6 mice before and after in vitro activation. qPCR was performed with Gapdh as the internal reference. Results were represented as 2 -ΔCt . **, p < 0.01. B) Competitive binding test revealed a hierarchical order of adenosine capture by immune cells as γδ T cells >> αβ T cells >> DCs. αβ responder T cells were isolated from IRBP 1-20 -immunized TCR-δ -/- mice. DCs were from 5 d GM-CSF-cultured bone marrow cells. In a 24-well plate the T cells were seeded at 1 x 10 6 /well and DCs at 5 x 10 5 /well. After incubation with1,000 nM H 3 -labeled adenosine for 1 h, the cells were harvested, and cell-bound H 3 -adenosine was counted by liquid scintillation. ** p < 0.01. C) Comparison of adenosine binding of γδ T cells isolated from naïve and immunized mice γδ T cells from immunized or naïve B6 mice, respectively. 1 x 10 5 /well cells were cultured in 96-well plate for 48h. 0–12,000 nM H 3 -labeled adenosine for 1 h, then the cells were harvested and cell-bound H 3 -adenosine counted by liquid scintillation. D) A2AR +/+ γδ T cells, but not A2AR -/- γδ T cells, possess an enhancing effect, which converted the inhibitory effect to enhancing on the αβ T cell response of an A2AR agonist. αβ and γδ T cells (1 x 10 5 ) were isolated from IRBP 1-20 -immunized TCR-δ -/- mice. The responder αβ T cells were labeled with CFSE and incubated for 5 d with immunizing peptide in the presence of APCs alone (A) or in the presence of the A2AR +/+ or A2AR -/- γδ T cells (B), in the absence or presence of an A2AR agonist, and with or without an addition of A2AR +/+ γδ T cells. The results shown are representative of three experiments.

Article Snippet: PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro, Activation Assay, Binding Assay, Cell Culture, Incubation, Labeling, Comparison

Journal: PLoS ONE

Article Title: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

doi: 10.1371/journal.pone.0199601

Figure Lengend Snippet: The enhancing effect of A2AR +/+ , A2AR -/- , and CD73 -/- γδ T cells were compared for promoting cytokine (IL-17) production of responder T cells obtained form TCR-δ -/- mice. Responder T cells were stimulated with the immunizing antigen and APCs in the absence or presence of indicated γδ T cells. The 48h cultured cell supernatants were sampled for testing IL-17 production by ELISA.

Article Snippet: PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

doi: 10.1371/journal.pone.0199601

Figure Lengend Snippet: Proliferative responses of CFSE-labeled αβ T cells isolated from immunized TCR-δ -/- B6 mice (1 x 10 5 /well) were assessed after in vitro incubation for 1 h with immunizing peptide and APCs alone (upper panels) or in the presence of 10 μM AMP (lower panels), 5% A2AR +/+ γδ T cells (Middle panels), or 5% A2AR -/- γδ T cells (right panels). The results shown are representative of those obtained in three separate experiments.

Article Snippet: PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Labeling, Isolation, In Vitro, Incubation

Journal: PLoS ONE

Article Title: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

doi: 10.1371/journal.pone.0199601

Figure Lengend Snippet: A) Failure to express A2AR rendered γδ T cells less activated. In a 24-well plate, 2 x 10 5 A2AR +/+ or A2AR -/- γδ T cells were incubated with medium only (Control), with pooled cytokines (IL-1, IL-7, and IL-23), and with or without or NECA (300 nM) or the A2AR-specific agonist CGS21680. At 48 h after the culture, IL-17 amounts in the culture supernatants were assessed by ELISA. B) Blockade of A2AR expression prevented γδ T cell activation. Surface staining for the T cell activation marker CD25 showing that A2AR antagonist treated A2AR +/+ γδ T cells express low amounts of CD25 after exposure to cytokines. The results shown are from a single experiment and are representative of those obtained in >5 experiments. ** , p < 0.01.

Article Snippet: PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Expressing, Activation Assay, Staining, Marker

Journal: Neuropharmacology

Article Title: Adenosine receptors as markers of brain iron deficiency: Implications for Restless Legs Syndrome

doi: 10.1016/j.neuropharm.2016.09.002

Figure Lengend Snippet: A–D. A2AR (A), D2R (B) and A1R (D) density in striatal tissue and A1R density in cortical tissue (C) in mice after 22 weeks of severe (3-ppm) or mild (7-ppm) ID-diet or control diet. Results are means ± S.E.M. of 6–12 animals/group. *, **: p<0.05 and p<0.01 compared to the control group, respectively; one-way ANOVA followed by Newman-Keul’s posthoc test. Bottom images show representative Western blots.

Article Snippet: Primary antibodies were: mouse monoclonal anti-TFR (1:1000 dilution; Invitrogen), mouse monoclonal anti-A2AR (1:1000 dilution; Upstate, Lake Placid, NY), rabbit polyclonal anti-D2R (1:1000 dilution, EMD Millipore, Darmstadt, Germany) and rabbit polyclonal anti-A1R (1:1000 dilution; Thermo Scientific; Wilmington, Laurel, DE).

Techniques: Western Blot

Journal: Neuropharmacology

Article Title: Adenosine receptors as markers of brain iron deficiency: Implications for Restless Legs Syndrome

doi: 10.1016/j.neuropharm.2016.09.002

Figure Lengend Snippet: A–D. Representative saturation experiments with the A1R antagonist [3H]DPCPX in cortical (A) and striatal (B) rat tissue and with the A2AR antagonist [3H]ZM241385 (C) and the D2R antagonist [3H]raclopride (D) in rat striatum after 3 weeks of a severe ID diet (3 ppm) and controls. E. Bmax values from all saturation experiments; results are means ± S.E.M. of 3–5 animals/group. *, **: p<0.05 and p<0.01 compared to the control group, respectively; non-paired t test.

Article Snippet: Primary antibodies were: mouse monoclonal anti-TFR (1:1000 dilution; Invitrogen), mouse monoclonal anti-A2AR (1:1000 dilution; Upstate, Lake Placid, NY), rabbit polyclonal anti-D2R (1:1000 dilution, EMD Millipore, Darmstadt, Germany) and rabbit polyclonal anti-A1R (1:1000 dilution; Thermo Scientific; Wilmington, Laurel, DE).

Techniques:

Journal: Neuropharmacology

Article Title: Adenosine receptors as markers of brain iron deficiency: Implications for Restless Legs Syndrome

doi: 10.1016/j.neuropharm.2016.09.002

Figure Lengend Snippet: A. Average locomotor activity in mice during 60 min after systemic (i.p.) administration of the A2AR antagonist KW-6002 (1 mg/kg), the A1R antagonist CPT (4.8 mg/kg) or caffeine (10 mg/kg). Broken line indicates the average level of locomotor activity at the end of the habituation period, which did not differ among the three groups. Results represent accumulative ambulatory counts and are expressed as means ± S.E.M. (12 animals per group). All mice were tested with the three drugs (with a counterbalanced schedule with 72 h of washout between each treatment) by the end of a 22-weeks period of severe (3-ppm) or mild (7-ppm) ID-diet or control diet. **: p<0.01 compared to the control group; one-way ANOVA followed by Newman-Keul’s posthoc test. B. Average locomotor activity in rats during 60 min after systemic (i.p.) administration of the CPT (4.8 mg/kg) or caffeine (10 mg/kg). Broken line indicates the average level of locomotor activity at the end of the habituation period, which did not differ among the three groups. Results represent accumulative ambulatory counts and are expressed are means ± S.E.M. (6 animals per group). Rats were only used once, for one of the treatments.

Article Snippet: Primary antibodies were: mouse monoclonal anti-TFR (1:1000 dilution; Invitrogen), mouse monoclonal anti-A2AR (1:1000 dilution; Upstate, Lake Placid, NY), rabbit polyclonal anti-D2R (1:1000 dilution, EMD Millipore, Darmstadt, Germany) and rabbit polyclonal anti-A1R (1:1000 dilution; Thermo Scientific; Wilmington, Laurel, DE).

Techniques: Activity Assay