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: Effects of dopamine (DA) microinjection or bath application on intracellular free calcium concentrations [iCa 2+ ] in hDRD5 receptor-expressing cells. (A) . Human U2-OS osteosarcoma cells natively expressing DRD1 and PMAT, but not DRD5, were pre-transfected with GFP-fluorescent human DRD5 receptor (green GFP-D5 fluorescence). Dopamine and other treatments were microinjected into the cell Soma followed by continuous 0.25 Hz recording of [iCa 2+ ] signal as indicated by Fura-2AM. fluorescence. Pictograms of [iCa 2+ ] from respective representative cells are shown (at least 12 transfected cells were tested with similar outcomes, and average results from 12 cells are shown in part D below). The control cell tested in parallel (top panel) was microinjected with buffered media alone, while the DA injected cell received 10 nM final intracellular concentration of DA. The [iCa 2+ ] signal integrated over a 6 min observation window as well as point observations at 2 and 6 min, are shown. (B) . Effects of co-microinjection of SCH23390 (SCH) 10 nM with dopamine 10 nM (Top panel) or of quinpirole microinjection (up to 10 μM, bottom panel) on [iCa 2+ ] response. SCH23390 blocked the iCa 2+ response to dopamine, while the D 2 agonist quinpirole (Quin) was without effect. (C) . Pictograms from representative experiments testing the effects of bath-applied DA on iCa 2+ mobilization in U2-OS cells. Bath application of 10 nM dopamine had no effect (Top panel), while 10 µM dopamine monitored over 11 min revealed a slowly rising wave of [iCa 2+ ] response that was highest toward the latest 11 min time point observed (Bottom panel). (D) . Quantified observations and temporal patterns of responses for microinjected versus bath applied dopamine. Continually recorded [iCa 2+ ] data resampled at 0.133 min intervals from 12 experiments are shown for microinjected DA 10 nM (Left graph, peak effect = 416%) in contrast with the effects of bath-applied DA 10 nM (Middle graph) and bath-applied DA 10 µM (Right graph). For bath-applied DA 10 μM, the average of the three observations made in the last 12 s of recording (106.2%, N = 12) was compared to the average of the three observations made in the first 12 s (0.2 min) of recording as the baseline (100%, N = 12); the comparison indicated a statistically significant increase in bath-applied dopamine-stimulated [iCa 2+ ] response (paired t -test, p = 0.019). The experimental system could not support extension of observation period past the 11th minute. (E) Expression indications for various dopamine receptors and PMAT in U2-OS cells. While D 1 and D 2 receptors as well as PMAT were expressed, there was no indication of D 5 receptor expression in the U2-OS cells.

Article Snippet: U2-OS cells were transfected with GFP-tagged human D 5 receptor (DRD5) cDNA ( OriGene.com , #RG202502) or pCMV6-AC-mGFP vector ( OriGene.com #PS100040) using Turbofectin-8 transfection reagent ( Origene.com #TF81001) according to manufacturer recommendations.

Techniques: Microinjection, Expressing, Transfection, Fluorescence, Control, Injection, Concentration Assay, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: Cortical dopamine D5 receptors regulate neuronal circuit oscillatory activity and memory in rats

doi: 10.1111/cns.14210

Figure Lengend Snippet: Knockdown of the D5R in PFC increases GSK‐3β activity. (A) The experimental timeline is shown. (B) Electrode placements in the PFC, OFC, thalamus, and HIP. (C) Representative images and quantification of fluorescence showing reduced PFC expression of the D5R (top panels) and GSK‐3β phosphorylation at Ser9 (bottom panels) following D5R‐shRNA induced knockdown. N = 7 rats/group (D) Images showing drd5 gene expression in control (left panels) animals or following drd5 mRNA knockdown (right panels). (E) Quantification of the number of drd5 mRNA expressing cells. N = 3 rats/group, 2 slices/rat. Quantified data are expressed as percent control. ** p < 0.01, *** p < 0.001 Student's t test.

Article Snippet: Subsequently, adjacent slices were incubated in primary antibodies rabbit anti‐pGSK‐3β (Ser9) (catalogue #ab9107166, 1:200, Abcam) or rabbit anti‐D5R (catalogue #ADR‐005, 1:200, Alomone Labs) for 60 h at 4°C.

Techniques: Knockdown, Activity Assay, Fluorescence, Expressing, shRNA, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Cortical dopamine D5 receptors regulate neuronal circuit oscillatory activity and memory in rats

doi: 10.1111/cns.14210

Figure Lengend Snippet: Effect of PFC D5R knockdown on oscillatory power activity in rats. Effect of PFC D5R knockdown on oscillatory power activity in rats. (A–D) Power spectra (left and center panels) and quantification of power (right panels) are shown. PFC D5R knockdown had the following regional effects on spectral power, (A) increased theta power in PFC, (B) increased theta power in OFC, and (C) increased theta power in HIP. (D) No effects of PFC D5R knockdown were evident in spectral power in the thalamus. Power curves are presented as normalized data with jackknife estimates of SEM shown as shaded areas. Quantified data are expressed as percent control. N = 7–8 rats/group, 1–2 electrodes/region/rat. * p < 0.05 Student's t test.

Article Snippet: Subsequently, adjacent slices were incubated in primary antibodies rabbit anti‐pGSK‐3β (Ser9) (catalogue #ab9107166, 1:200, Abcam) or rabbit anti‐D5R (catalogue #ADR‐005, 1:200, Alomone Labs) for 60 h at 4°C.

Techniques: Knockdown, Activity Assay, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Cortical dopamine D5 receptors regulate neuronal circuit oscillatory activity and memory in rats

doi: 10.1111/cns.14210

Figure Lengend Snippet: Effect of PFC D5R knockdown on oscillatory coherence in rats. (A–F) Coherence spectra (left panels) and quantification (right panels) are shown. (A) PFC D5R knockdown increased PFC‐OFC theta coherence, and (B) reduced PFC–thalamus high gamma coherence. (C–F) No significant effects on coherence were observed between PFC–HIP, OFC‐HIP, OFC‐thalamus, or thalamus‐HIP. Coherence curves are presented with jackknife estimates of SEM shown as shaded areas. Quantified data are expressed as percent control. N = 7–8 rats/group, 1–2 electrodes/region/rat. * p < 0.05, ** p < 0.01 Student's t test.

Article Snippet: Subsequently, adjacent slices were incubated in primary antibodies rabbit anti‐pGSK‐3β (Ser9) (catalogue #ab9107166, 1:200, Abcam) or rabbit anti‐D5R (catalogue #ADR‐005, 1:200, Alomone Labs) for 60 h at 4°C.

Techniques: Knockdown, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Cortical dopamine D5 receptors regulate neuronal circuit oscillatory activity and memory in rats

doi: 10.1111/cns.14210

Figure Lengend Snippet: D5R knockdown in PFC induces deficits in learning and memory. (A) D5R knockdown in PFC induced deficits in object recognition memory in the NOR test. (B) Impaired spatial memory in the OL test was also evident. (C) D5R knockdown also induced impairment in associative recognition memory when tested in the OiP. (D) In the Y‐maze, D5R knockdown had no effects on short‐term recognition memory (left panel) but resulted in impairment on long‐term memory (right panel). N = 7–8 rats/group, * p < 0.05, ** p < 0.01, *** p < 0.001 Student's t test.

Article Snippet: Subsequently, adjacent slices were incubated in primary antibodies rabbit anti‐pGSK‐3β (Ser9) (catalogue #ab9107166, 1:200, Abcam) or rabbit anti‐D5R (catalogue #ADR‐005, 1:200, Alomone Labs) for 60 h at 4°C.

Techniques: Knockdown