Journal: Brain research bulletin

Article Title: Increased DNA methylation of Igf2 in the male hippocampus regulates age-related deficits in synaptic plasticity and memory

doi: 10.1016/j.brainresbull.2025.111509

Figure Lengend Snippet: ( A,B ) Effectiveness of developed CRISPR guide RNAs (gRNAs) against the promoter region of Igf2 variants was tested in rat B35 cells with a dCas9-VPR transcriptional activator plasmid. qRT-PCR analysis revealed a successful increase in Igf2 variant 1 and 2 expression. ( C ) Data comes from a subset of the behaviorally tested animals presented in E-F below. Igf2 -gRNA in combination with dCas9-TET1 were bilaterally infused into the CA1 area of the dorsal hippocampus. Four weeks later, DNA was collected and processed with 5-hydroxymethylation(5-hmC) immunoprecipitation assay (hmeDIP). dCas9-TET1 resulted in an increase in DNA 5-hmC levels at the Igf2 promoter. ( D ) Male aged (22-m.o.) rats received bilateral injections of either Igf2 -gRNA alone or in combination with dCas9-TET1 into the CA1 area of the dorsal hippocampus. Four weeks later, animals were trained in contextual fear conditioning and re-exposed to the training context 24 h later to assess memory retention (n = 7–11 per group). ( E ) The dCas9-TET1 manipulation did not impact performance during training. ( F ) During testing, aged rats injected with dCas9-TET1 targeting Igf2 had significantly increased memory retention. * P < 0.05 from Control.

Article Snippet: Rat B35 neuroblastoma cell line (#CRL-2754; ATCC, Manassas, VA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (#30–2002; ATCC) supplemented with 10 % Fetal Bovine serum (#35–016-CV; Corning, Corning, NY) and 0.1 % Penicillin/Streptomycin (#15070063; Gibco, Gaitherburg, MD).

Techniques: CRISPR, Plasmid Preparation, Quantitative RT-PCR, Variant Assay, Expressing, Immunoprecipitation, Injection, Control

Journal: Frontiers in Pharmacology

Article Title: Dopamine internalization via Uptake 2 and stimulation of intracellular D 5 -receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

doi: 10.3389/fphar.2024.1422998

Figure Lengend Snippet: Dopamine agonist effects on CDP-diacylglycerol accumulation in cultured brain slices and neural cells that lack presynaptic dopamine terminals or endogenous dopamine. (A) . CDP-diacylglycerol accumulation in [ 3 H]cytidine-prelabeled organotypic striatal slices following 90 min incubations with various concentrations of dopamine (N = 8) or the D 1 -like receptor agonists SKF38393 (N = 8) and SKF83959 (N = 4). [ 3 H]CDP-diacylglycerol responses were converted to percentages relative to Control (608 ± 121 dpm/mg protein, N = 20) to yield the mean ± SD values shown. Each drug concentration-dependently increased [ 3 H]CDP-diacylglycerol in the cultured slice preparations as compared by Two-Way ANOVA, p < 0.001 compared across drug concentrations; p < 0.01 compared among drugs. Insert micrograph illustrates the ordinary appearance of a MAP2-stained 7 day-cultured organotypic striatal slice that has thinned out to reveal networks of neurons within the organotypic matrix. (B) . Plot of data as net increase above control percent maximal response for each agent, thus the control response equals 0% and the maximal response equals 100%. A sigmoidal concentration-response function was fitted to the data to yield the fit curves shown. From this, pharmacological indices of potency (EC 50 ) and efficacy (E max ) were determined for each drug using GraphPad Prism. Insert shows the computed geometric mean EC 50 and E max values with 95% confidence intervals (95% CI) summarized for the test drugs. (C) . Agonist effects on [ 3 H]CDP-diacylglycerol accumulation in frontal cortical neurons incubated with [ 3 H]cytidine for 60 min. Each bar is mean ± SD for dopamine (N = 9) or SKF38393 (N = 6) as shown. Two-Way ANOVA analysis indicated differences between the effects of dopamine and SKF38393 ( p < 0.05) as well as significant concentration-dependent effects of the drugs ( p < 0.0001); subsequent posthoc tests for each drug showed significantly different mean effects at the 30–100 µM concentrations of dopamine or SKF38393. * p < 0.05; **** p < 0.0001 compared to respective control (0 µM) responses by Dunnett tests. (D) Effects of dopamine on CDP-diacylglycerol accumulation in B35 neuroblastoma cells incubated with [ 3 H]cytidine and the indicated dopamine concentrations for 60 min. Each bar is the mean ± SD (N = 6). Drug concentrations of 30 µM and higher produced statistically significant increases in nucleolipid formation based on One-Way ANOVA analysis ( p < 0.0001). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the baseline (0 µM dopamine) group by posthoc Dunnett tests.

Article Snippet: Rat embryonic cortex-derived B35 neuroblastoma cells were purchased and authenticated from mycoplasma-free stock of American Type Culture Collection ( ATCC.org ) and maintained in DMEM media (Life Technologies/ThermoFisher.com ) supplemented with 10% fetal bovine serum, FBS (Atlanta Biological, bio-techne.com ) under an atmosphere of 5% CO 2 and 37°C.

Techniques: Cell Culture, Control, Concentration Assay, Staining, Incubation, Produced

Journal: Frontiers in Pharmacology

Article Title: Dopamine internalization via Uptake 2 and stimulation of intracellular D 5 -receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

doi: 10.3389/fphar.2024.1422998

Figure Lengend Snippet: Intracellular dopamine (DA) uptake and effects of competing ligands and Uptake 2 modulation. (A) . Rat B35 cortical neuroblastoma cells demonstrably expressing PMAT (in addition to D 1 and D 5 receptors but not D 2 receptor) were used. (B) . Fluorescent dansyl-labeled DA uptake in B35 cells incubated for 30 min with 30 µM dansyl-dopamine only (Left) or 30 µM dansyl-dopamine in the presence of 10 µM decynium-22 (Right). Cells were visualized under confocal microscopy. Intracellular green fluorescence indicates dansyl-dopamine entry and retention in the B35 cells. Note the discrete intracellular distribution of the dopamine fluorescence in incubations with dansyl-dopamine, especially in the absence of decynium-22. Arrows show: Dansyldopamine fluorescence in cell membrane (White arrowhead); Cellular subregion of low dansyldopamine fluorescence (Blue arrow); Cellular subregion of high dansyldopamine fluorescence (Red arrow). (C) . Cellular [ 3 H]DA uptake in B35 cells incubated with indicated molar concentrations of total (cold + radiolabeled) dopamine in the presence or absence of 10 µM decynium-22 (D22). Nonspecific uptake was defined with 30 µM D22. Each bar is the mean ± SD (N = 7–9). Two-Way ANOVA showed significant effects of dopamine concentration ( p < 0.0001) and of D22 pretreatment ( p < 0.0001) on [ 3 H]dopamine uptake. * p < 0.001 compared to the paired concentration of dopamine alone. (D) . Effects on [ 3 H]DA uptake of Uptake 2 inhibitors D22 and corticosterone (Top) and competition by DA ligands (Bottom). Indicated concentrations of each uptake inhibitor compound were tested against 100 µM [ 3 H]DA in B35 cells. Nonspecific uptake was defined with 300 µM SKF38393. Each point is the mean ± sem (N = 6). Two-Way ANOVA was performed for the competing agents followed by Dunnett testing to compare the concentration effect of each agent to its control ([ 3 H]DA alone). * p < 0.05, *** p < 0.001 compared to Control (Con [ 3 H]DA alone) for each drug treatment by Dunnett test.

Article Snippet: Rat embryonic cortex-derived B35 neuroblastoma cells were purchased and authenticated from mycoplasma-free stock of American Type Culture Collection ( ATCC.org ) and maintained in DMEM media (Life Technologies/ThermoFisher.com ) supplemented with 10% fetal bovine serum, FBS (Atlanta Biological, bio-techne.com ) under an atmosphere of 5% CO 2 and 37°C.

Techniques: Expressing, Labeling, Incubation, Confocal Microscopy, Fluorescence, Membrane, Concentration Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Dopamine internalization via Uptake 2 and stimulation of intracellular D 5 -receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

doi: 10.3389/fphar.2024.1422998

Figure Lengend Snippet: Dopamine agonist effects on CDP-diacylglycerol accumulation in cultured brain slices and neural cells that lack presynaptic dopamine terminals or endogenous dopamine. (A) . CDP-diacylglycerol accumulation in [ 3 H]cytidine-prelabeled organotypic striatal slices following 90 min incubations with various concentrations of dopamine (N = 8) or the D 1 -like receptor agonists SKF38393 (N = 8) and SKF83959 (N = 4). [ 3 H]CDP-diacylglycerol responses were converted to percentages relative to Control (608 ± 121 dpm/mg protein, N = 20) to yield the mean ± SD values shown. Each drug concentration-dependently increased [ 3 H]CDP-diacylglycerol in the cultured slice preparations as compared by Two-Way ANOVA, p < 0.001 compared across drug concentrations; p < 0.01 compared among drugs. Insert micrograph illustrates the ordinary appearance of a MAP2-stained 7 day-cultured organotypic striatal slice that has thinned out to reveal networks of neurons within the organotypic matrix. (B) . Plot of data as net increase above control percent maximal response for each agent, thus the control response equals 0% and the maximal response equals 100%. A sigmoidal concentration-response function was fitted to the data to yield the fit curves shown. From this, pharmacological indices of potency (EC 50 ) and efficacy (E max ) were determined for each drug using GraphPad Prism. Insert shows the computed geometric mean EC 50 and E max values with 95% confidence intervals (95% CI) summarized for the test drugs. (C) . Agonist effects on [ 3 H]CDP-diacylglycerol accumulation in frontal cortical neurons incubated with [ 3 H]cytidine for 60 min. Each bar is mean ± SD for dopamine (N = 9) or SKF38393 (N = 6) as shown. Two-Way ANOVA analysis indicated differences between the effects of dopamine and SKF38393 ( p < 0.05) as well as significant concentration-dependent effects of the drugs ( p < 0.0001); subsequent posthoc tests for each drug showed significantly different mean effects at the 30–100 µM concentrations of dopamine or SKF38393. * p < 0.05; **** p < 0.0001 compared to respective control (0 µM) responses by Dunnett tests. (D) Effects of dopamine on CDP-diacylglycerol accumulation in B35 neuroblastoma cells incubated with [ 3 H]cytidine and the indicated dopamine concentrations for 60 min. Each bar is the mean ± SD (N = 6). Drug concentrations of 30 µM and higher produced statistically significant increases in nucleolipid formation based on One-Way ANOVA analysis ( p < 0.0001). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the baseline (0 µM dopamine) group by posthoc Dunnett tests.

Article Snippet: Rat embryonic cortex-derived B35 neuroblastoma cells were purchased and authenticated from mycoplasma-free stock of American Type Culture Collection ( ATCC.org ) and maintained in DMEM media (Life Technologies/ThermoFisher.com ) supplemented with 10% fetal bovine serum, FBS (Atlanta Biological, bio-techne.com ) under an atmosphere of 5% CO 2 and 37°C.

Techniques: Cell Culture, Control, Concentration Assay, Staining, Incubation, Produced

Journal: Frontiers in Pharmacology

Article Title: Dopamine internalization via Uptake 2 and stimulation of intracellular D 5 -receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

doi: 10.3389/fphar.2024.1422998

Figure Lengend Snippet: Intracellular dopamine (DA) uptake and effects of competing ligands and Uptake 2 modulation. (A) . Rat B35 cortical neuroblastoma cells demonstrably expressing PMAT (in addition to D 1 and D 5 receptors but not D 2 receptor) were used. (B) . Fluorescent dansyl-labeled DA uptake in B35 cells incubated for 30 min with 30 µM dansyl-dopamine only (Left) or 30 µM dansyl-dopamine in the presence of 10 µM decynium-22 (Right). Cells were visualized under confocal microscopy. Intracellular green fluorescence indicates dansyl-dopamine entry and retention in the B35 cells. Note the discrete intracellular distribution of the dopamine fluorescence in incubations with dansyl-dopamine, especially in the absence of decynium-22. Arrows show: Dansyldopamine fluorescence in cell membrane (White arrowhead); Cellular subregion of low dansyldopamine fluorescence (Blue arrow); Cellular subregion of high dansyldopamine fluorescence (Red arrow). (C) . Cellular [ 3 H]DA uptake in B35 cells incubated with indicated molar concentrations of total (cold + radiolabeled) dopamine in the presence or absence of 10 µM decynium-22 (D22). Nonspecific uptake was defined with 30 µM D22. Each bar is the mean ± SD (N = 7–9). Two-Way ANOVA showed significant effects of dopamine concentration ( p < 0.0001) and of D22 pretreatment ( p < 0.0001) on [ 3 H]dopamine uptake. * p < 0.001 compared to the paired concentration of dopamine alone. (D) . Effects on [ 3 H]DA uptake of Uptake 2 inhibitors D22 and corticosterone (Top) and competition by DA ligands (Bottom). Indicated concentrations of each uptake inhibitor compound were tested against 100 µM [ 3 H]DA in B35 cells. Nonspecific uptake was defined with 300 µM SKF38393. Each point is the mean ± sem (N = 6). Two-Way ANOVA was performed for the competing agents followed by Dunnett testing to compare the concentration effect of each agent to its control ([ 3 H]DA alone). * p < 0.05, *** p < 0.001 compared to Control (Con [ 3 H]DA alone) for each drug treatment by Dunnett test.

Article Snippet: Rat embryonic cortex-derived B35 neuroblastoma cells were purchased and authenticated from mycoplasma-free stock of American Type Culture Collection ( ATCC.org ) and maintained in DMEM media (Life Technologies/ThermoFisher.com ) supplemented with 10% fetal bovine serum, FBS (Atlanta Biological, bio-techne.com ) under an atmosphere of 5% CO 2 and 37°C.

Techniques: Expressing, Labeling, Incubation, Confocal Microscopy, Fluorescence, Membrane, Concentration Assay, Control