Journal: The Journal of biological chemistry

Article Title: GABA A α 6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer *

doi: 10.1074/jbc.M700111200

Figure Lengend Snippet: Full saturation [3H]Ro15-4513 binding curves were generated using +/+ and stg cerebellar membranes and a concentration range of 0.3–40 nm [3H]Ro15-4513. Nonspecific binding was determined in the presence of Ro15-1788 (10 μm). [3H]Ro15-4513 binding in the presence of flunitrazepam (10 μm), defined BZ-IS-binding sites, and hence benzodiazepine-sensitive (BZ-S) sites could be determined by subtraction of the BZ-IS-binding sites from total [3H]Ro15-4513 specific binding sites. The bar graph illustrates the difference in Bmax values calculated for [3H]Ro15-4513 binding to +/+ and stg membranes. Results show differences in Bmax for total specific binding (100 ± 5% and 81 ± 7%), BZ-S binding (100 ± 11% and 92 ± 9%), and BZ-IS binding (100 ± 3% and 57 ± 12%) in +/+ and stg membranes, respectively. The table summarizes the estimated Bmax and KD values for [3H]Ro15-4513 binding to +/+ and stg cerebellar membranes. Data are representative of the mean ± S.E. for assays conducted in triplicate on two separate membrane preparations per mouse strain (n = 6). Ten cerebella were used for each preparation per mouse strain.

Article Snippet: [ 3 H]Muscimol and [ 3 H]Ro15-4513 were purchased from PerkinElmer Life Sciences.

Techniques: Binding Assay, Generated, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: GABA A α 6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer *

doi: 10.1074/jbc.M700111200

Figure Lengend Snippet: A, total [3H]Ro15-4513 binding. Panel i, in situ autoradiography of total [3H]Ro15-4513 binding (γ2-containing GABARs) to +/+ and stg cerebella using a near-saturating concentration of [3H]Ro15-4513 (20 nm). Nonspecific binding was determined by competitive displacement of Ro15-4513 with the benzodiazepine receptor antagonist, Ro15-1788 (10 μm). The signal obtained under these latter conditions was at the level of film background. Two representative comparable sections per mouse strain are shown. Panel ii, magnified image of cerebellar lobules labeled with [3H]Ro15-4513. Panel iii, histogram illustrating the results of quantitative image analysis of grayscale intensities using image J software to determine the relative amounts of ligand bound in the cerebellar granule cell layers. A small (10 ± 4%) but insignificant (p > 0.05) reduction in total [3H]Ro15-4513 binding in the cerebellar granule cell layer of stg was determined. Data shown are representative of at least three +/+ and three stg brains and a minimum of 10 sections per brain. B, BZ-IS [3H]Ro15-4513 binding. Panel i, in situ autoradiography of flunitrazepam (10 μm)-insensitive [3H]Ro15-4513 (BZ-ISR) binding (α6βγ2, α1α6βγ2 GABARs) in +/+ and stg cerebella using a saturating concentration of [3H]Ro15-4513 (20 nm). Nonspecific binding was determined by competitive displacement of Ro15-4513 with benzodiazepine receptor antagonist, Ro15-1788 (10 μm). The signal obtained under these conditions was at the level of film background. Panel ii, magnified image of cerebellar lobules following autoradiography to identify BZ-ISRs. Panel iii, histogram illustrating the results of quantitative image analysis of grayscale intensities using image J software to determine the relative amounts of radioligand bound in the cerebellar granule cell layer. A small but significant reduction (by 21 ± 7%; p < 0.05) in flunitrazepam-insensitive [3H]Ro15-4513 binding in the cerebellar granule cell layer of stg was determined. Data shown are representative of at least three +/+ and three stg brains and a minimum of 10 sections per brain. C, BZ-S [3H]flunitrazepam binding. Panel i, in situ autoradiography of [3H]flunitrazepam (BZ-SR) binding (e.g. α1βγ2 GABARs) in +/+ and stg cerebella using [3H]flunitrazepam (5 nm). Nonspecific binding was determined by competitive displacement of flunitrazepam with benzodiazepine receptor antagonist, Ro15-1788 (10 μm). The signal obtained under these conditions was at the level of film background. Panel ii, magnified image of cerebellar lobules following autoradiography to identify BZ-SRs. Panel iii, histogram illustrating the results of quantitative image analysis of grayscale intensities using image J software to determine the relative amounts of radioligand bound in the cerebellar granule cell layer. A small, nonsignificant increase (by 13 ± 6%; p > 0.05) in [3H]flunitrazepam binding in the cerebellum of stg was determined.

Article Snippet: [ 3 H]Muscimol and [ 3 H]Ro15-4513 were purchased from PerkinElmer Life Sciences.

Techniques: Binding Assay, In Situ, Autoradiography, Concentration Assay, Labeling, Software

Journal: The Journal of biological chemistry

Article Title: GABA A α 6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer *

doi: 10.1074/jbc.M700111200

Figure Lengend Snippet: Zolpidem (1 nm to 10 μm) was used to competitively displace BZ-S [3H]Ro15-4513 (5 nm) equilibrium binding from cerebellar membrane homogenates derived from adult +/+ and stg mice. [3H]Ro15-4513 binding displaced by flunitrazepam (10 μm) defined benzodiazepine-sensitive (BZ-S) sites. 100% binding was that obtained in the absence of competitive ligand.

Article Snippet: [ 3 H]Muscimol and [ 3 H]Ro15-4513 were purchased from PerkinElmer Life Sciences.

Techniques: Binding Assay, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: α4-Containing GABA A Receptors are Required for Antagonism of Ethanol-Induced Motor Incoordination and Hypnosis by the Imidazobenzodiazepine Ro15-4513

doi: 10.3389/fphar.2011.00018

Figure Lengend Snippet: Ro15-4513 antagonism of ethanol-induced motor ataxia . (A) Performance of WT and GABA A -Rα4 subunit KO mice on fixed speed (8 rpm) rotarod following pretreatment with 2 g/kg ethanol 5 min prior to treatment with 10 mg/kg Ro15-4513 or vehicle.* p ≤ 0.01 – WT ethanol/vehicle vs WT ethanol/Ro15-4513. (B) AUCs of response curves in (A) . Effect of genotype, treatment, and the interaction were not significant. (C) Performance on fixed speed (12 rpm) rotarod following treatment with 1.5 g/kg ethanol ±3 mg/kg Ro15-4513. * p < 0.005, ** p < 0.0001 – WT ethanol vs WT ethanol + Ro15-4513. (D) AUCs of response curves in (C) . WT and KO mice did not differ following injection of ethanol alone. Ro15-4513 antagonized ethanol-induced motor ataxia in WT, but not in KO mice. All values are expressed as mean ± SEM.

Article Snippet: Ro15-4513 was freshly prepared in 0.3% Tween-80 in saline at 1 mg/ml on the morning of the experiment in BD Falcon round bottomed tubes.

Techniques: Injection

Journal: Frontiers in Pharmacology

Article Title: α4-Containing GABA A Receptors are Required for Antagonism of Ethanol-Induced Motor Incoordination and Hypnosis by the Imidazobenzodiazepine Ro15-4513

doi: 10.3389/fphar.2011.00018

Figure Lengend Snippet: Antagonism of ethanol (3. 5 g/kg)-induced LORR by Ro15-4513 (20 mg/kg) . (A) In male mice, Ro15-4513 antagonized the duration of ethanol-induced LORR in WT, but not in KO. (B) In female mice, Ro15-4513 antagonized ethanol-induced LORR ( p < 0.0001) irrespective of genotype. All values are expressed as mean ± SEM.

Article Snippet: Ro15-4513 was freshly prepared in 0.3% Tween-80 in saline at 1 mg/ml on the morning of the experiment in BD Falcon round bottomed tubes.

Techniques: