Journal: Frontiers in Neural Circuits

Article Title: Differential Expression of Dopamine D5 Receptors across Neuronal Subtypes in Macaque Frontal Eye Field

doi: 10.3389/fncir.2018.00012

Figure Lengend Snippet: Information on primary antibodies used for immunofluorescence.

Article Snippet: We performed two controls to verify the specifity of the D5R antibody from Alomone Labs (ADR-005, see Supplementary Figure ).

Techniques: Immunofluorescence

Journal: Frontiers in Neural Circuits

Article Title: Differential Expression of Dopamine D5 Receptors across Neuronal Subtypes in Macaque Frontal Eye Field

doi: 10.3389/fncir.2018.00012

Figure Lengend Snippet: Distribution of D5Rs across cortical layers. (A) Number of NeuN+ neurons that co-express D5R for cortical layers I, II–III, IV, V and VI per mm 2 . (B) Proportion of NeuN+ neurons that express D5Rs for cortical layers I, II–III, IV, V and VI.

Article Snippet: We performed two controls to verify the specifity of the D5R antibody from Alomone Labs (ADR-005, see Supplementary Figure ).

Techniques:

Journal: Frontiers in Neural Circuits

Article Title: Differential Expression of Dopamine D5 Receptors across Neuronal Subtypes in Macaque Frontal Eye Field

doi: 10.3389/fncir.2018.00012

Figure Lengend Snippet: D5R expression on different cell types. (A) Co-expression of D5Rs (green) and SMI-32+ putative long-range projection pyramidal neurons (red; left) and co-expression of D5Rs with a general pyramidal neuron stain (neurogranin, red; right). (B) Proportion of frontal eye field (FEF) SMI-32+ and Neurogranin+ neurons that express D5Rs. (C) Expression of D5Rs (green) among different interneuron subtypes (red): parvalbumin+ (top left), calretinin+ (top right), calbindin+ (bottom left), somatostatin+ (bottom right). (D) Proportion of inhibitory interneurons that express D5Rs. Statistical comparisons were made between parvalbumin+, calbindin+ and calretinin+ neurons. Somatostatin+ neurons were excluded because they were too sparse to be used in statistical comparisons. For all panels: scale bar is equal to 100 μm and *** denotes significance at the level p ≤ 0.001.

Article Snippet: We performed two controls to verify the specifity of the D5R antibody from Alomone Labs (ADR-005, see Supplementary Figure ).

Techniques: Expressing, Staining

Journal: Frontiers in Neural Circuits

Article Title: Differential Expression of Dopamine D5 Receptors across Neuronal Subtypes in Macaque Frontal Eye Field

doi: 10.3389/fncir.2018.00012

Figure Lengend Snippet: Proportion of D5Rs on different cell types across cortical layers. (A) Neurogranin+ pyramidal neurons and SMI-32+ putative long-range projection neurons. (B) Parvalbumin+, calbindin+, calretinin+ and somatostatin+ inhibitory interneurons. Proportions are broken down by cortical layer (I, II–III, IV, V and VI) and compared to SMI-32+ D5R+ proportions (light gray bars). *, ** and *** indicate significance at the levels of p ≤ 0.05, 0.01 and 0.001 (Bonferonni-adjusted values), respectively. Differences in the proportion of D5R+ neurons across layers were also calculated individually for each cell type. Only calretinin+ neurons exhibited a significant difference in D5R+ proportions across layers: indicated with a vertical line spanning layers II-VI and significance at the level of p ≤ 0.001 (Bonferonni-adjusted value) is indicated by ††† .

Article Snippet: We performed two controls to verify the specifity of the D5R antibody from Alomone Labs (ADR-005, see Supplementary Figure ).

Techniques:

Journal: Frontiers in Neural Circuits

Article Title: Differential Expression of Dopamine D5 Receptors across Neuronal Subtypes in Macaque Frontal Eye Field

doi: 10.3389/fncir.2018.00012

Figure Lengend Snippet: Proportion of D5R+ neurons by cell type for different cortical layers. For cortical layers I, II–III, IV, V and VI: proportion of D5R+ cells that express a given cell type marker (SMI-32, neurogranin, parvalbumin, calretinin, somatostatin).

Article Snippet: We performed two controls to verify the specifity of the D5R antibody from Alomone Labs (ADR-005, see Supplementary Figure ).

Techniques: Marker

Journal: Immunology

Article Title: Instantaneous depolarization of T cells via dopamine receptors, and inhibition of activated T cells of Psoriasis patients and inflamed human skin, by D1‐like receptor agonist: Fenoldopam

doi: 10.1111/imm.13109

Figure Lengend Snippet: Selective D1/5R agonists induce depolarization of activated CD3+ normal human T cells within 15 seconds. (a–g) The fluorescence profiles of the voltage‐sensitive fluorescence dye DiBac3 inside activated normal human T cells, both before (gray) and then again 15 seconds after (colored) addition of either: Control buffer (PBS) only (a), D1R/D15R (D1‐like) agonists: SKF 38393 (b) and Fenoldopam (c); D2R agonist: Sumanirole (d); D3R agonists: 7‐OH DPAT (e) and PD 128907 (f); or D4R agonist: PD 168077 (g). All the DR agonists were used in a final concentration of 10−7 M (100 nM), prepared freshly before each experiment, from either their powders or very concentrated stocks stored at −80°. The corresponding intensity of the voltage‐sensitive fluorescence dye DiBac3 was determined by the geometric mean (GM) of the fluorescence (FL1), in each tube before and after addition of each agonist, and all the GMs and fold change/shift are shown in Table 1. As for the depolarization of resting T cells shown in Fig 2, the results of the activated T cells shown in the figure are of one representative experiment, of two performed altogether, on T cells of different healthy human paticipants. In both experiments the same pattern of results was observed, but the actual extent of the depolarization varied. This was not surprising in view of the differences between individuals with regard to the level of DRs on the cell surface of their T cells found in7 and also in the present study (data not shown).

Article Snippet: In single‐staining experiments, normal human CD3 + T cells (isolated from fresh human blood samples of healthy blood donors) were stained with rabbit‐anti‐human D1R, D2R, D3R, D4R and D5R polyclonal antibodies (Calbiochem, http://www.merckmillipore.com/ ) (1 : 50), or with rabbit isotype control (1 : 50), and then with fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit antibody (Jackson Laboratories, https://www.jacksonimmuno.com ) (1 : 100).

Techniques: Fluorescence, Concentration Assay

Journal: Immunology

Article Title: Instantaneous depolarization of T cells via dopamine receptors, and inhibition of activated T cells of Psoriasis patients and inflamed human skin, by D1‐like receptor agonist: Fenoldopam

doi: 10.1111/imm.13109

Figure Lengend Snippet: Selective D1/5R agonists induce depolarization of resting CD3+ normal human T cells within 15 seconds. (a–g) The fluorescence profiles of the voltage‐sensitive fluorescence dye DiBac3 inside resting normal human T cells, both before (gray) and then again 15 seconds after (colored) addition of either: Control buffer (PBS) only (a), D1R/D15R (D1‐like) agonists: SKF 38393 (b) and Fenoldopam (c); D2R agonist: Sumanirole (d); D3R agonists: 7‐OH DPAT (e) and PD 128907 (f); D4R agonist: PD 168077 (g). All the DR agonists were used in a final concentration of 10−7 M (100 nM), prepared freshly before each experiment, from either their powders or very concentrated stocks stored in at –80°. The corresponding intensity of the voltage‐sensitive fluorescence dye DiBac3 was determined by the geometric mean (GM) of the fluorescence (FL1), in each tube before and after addition of each agonist, and all the GMs and fold change/shift are shown in Table 1. The results shown in the figure are of one representative experiment, of two performed altogether, on T cells of different healthy human participants. In both experiments the same pattern of results was observed, but the actual extent of the depolarization varied. This was not surprising in view of the differences between individuals with regard to the level of DRs on the cell surface of their T cells (found in 7 and also in the current study (data not shown).

Article Snippet: In single‐staining experiments, normal human CD3 + T cells (isolated from fresh human blood samples of healthy blood donors) were stained with rabbit‐anti‐human D1R, D2R, D3R, D4R and D5R polyclonal antibodies (Calbiochem, http://www.merckmillipore.com/ ) (1 : 50), or with rabbit isotype control (1 : 50), and then with fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit antibody (Jackson Laboratories, https://www.jacksonimmuno.com ) (1 : 100).

Techniques: Fluorescence, Concentration Assay

Journal: Immunology

Article Title: Instantaneous depolarization of T cells via dopamine receptors, and inhibition of activated T cells of Psoriasis patients and inflamed human skin, by D1‐like receptor agonist: Fenoldopam

doi: 10.1111/imm.13109

Figure Lengend Snippet: Normal human CD3+ T cells express all types of dopamine receptors (DRS) – D1R‐D5R on their cell surface, and the level of D1R, D5R and D4R are higher in activated T cells than in resting T cells. (a–e; Upper Figs) Expression of D1R–D5R on resting normal human CD3+ T cells. (f–j; Lower Figs) Expression of D1R–D5R on CD3/CD28‐activated normal human CD3+ T cells. The colored graphs show the levels of immunofluorescence staining, first with rabbit‐anti‐human antibodies selective to each DR type, and then with a secondary‐FITC‐conjugated goat anti‐rabbit antibody. The control gray fluorescence profiles in each graph represent the control non‐specific staining, first with rabbit isotype control, and then with the same secondary antibody. The results are of one representative experiment, of two performed on T cells of different healthy human individuals. In both experiments the same pattern of results was observed, but the actual numbers/expression levels were different. This observation is in line with the findings of Kustrimovic et al.,7 that human T cells express all DR types, but that there is a remarkable variability between different people with regard to the exact level of each DR type expressed on the cell surface of their T cells.

Article Snippet: In single‐staining experiments, normal human CD3 + T cells (isolated from fresh human blood samples of healthy blood donors) were stained with rabbit‐anti‐human D1R, D2R, D3R, D4R and D5R polyclonal antibodies (Calbiochem, http://www.merckmillipore.com/ ) (1 : 50), or with rabbit isotype control (1 : 50), and then with fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit antibody (Jackson Laboratories, https://www.jacksonimmuno.com ) (1 : 100).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

doi: 10.1371/journal.pone.0025639

Figure Lengend Snippet: Responses are plotted as percent change from the baseline fEPSPs as a function of time. Numbers on the representative traces show the time on the graph at which they were recorded. A) SKF81297-induced LTP in the cocaine CPP group (clear triangles, 151.4±8.8%, * p <0.05, n = 6) is completely blocked by the PLD-linked mGluR antagonist (PCCG-13, filled triangles, 95.0±9.2%, n = 6). B) mGluR1 receptor antagonist (LY367385, filled triangles, 106.0±6.7%, n = 6) blocks the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). C) The mGluR5 antagonist (MPEP, filled triangles, 122.7±5.6%, n = 6) significantly reduces but does not abolish the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). D) PLC antagonist (U-73122, filled triangles, 128.2±6.1%, n = 6), reduces but does not eliminate the SKF81297-induced LTP (clear triangles, * p <0.05, n = 6). For comparison, panels A and B use same data graphs and fEPSP traces for the slices from cocaine CPP group superfused with SKF81297 as shown in , and .

Article Snippet: Primary antibodies (with the references provided where the antibodies were tested for specificity) included: metabotropic glutamate receptors [mGluR1 (AGC-006) and mGluR5 (AGC-007) ] from Alomone (Jerusalem, Israel); phospholipase D [PLD1 (sc-25512) and PLD2 (sc-25513), ] and actin (sc-1616) from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA); dopamine receptor D1R (AB20066) from Abcam (Cambridge, MA); D5R (MAB5292) from Millipore (Temecula, CA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clone 6C5) from Advanced Immunochemicals Inc. (Long Beach, CA).

Techniques:

Journal: PLoS ONE

Article Title: Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

doi: 10.1371/journal.pone.0025639

Figure Lengend Snippet: The dotted line indicates PLD activity associated with control slices (no EtOH added) which was determined for each animal and used to calculate the change in PLD activity levels with EtOH and/or drug application. Basal levels represent the increase in PLD activity observed in the EtOH-treated slices compared to the no EtOH controls; * p <0.05 compared to the corresponding saline control and # p <0.05 compared to the cocaine CPP group basal PLD activity. Basal PLD activity was significantly increased (*** p <0.001, n = 50) in the cocaine CPP group (dark bars, 527.3±94.3) compared to the saline-treated group (white bars, 142.6±36.9). SKF81297, the D1/5R agonist, application increased the basal levels in the cocaine CPP group significantly (1722.0±176.9, n = 12, # p <0.05) compared to the basal PLD activity observed with EtOH treatment alone in the same experimental group. The D1/5R antagonist, SCH23390, completely blocked basal PLD activity (91.2±21.9, n = 12, ## p <0.01) in the cocaine CPP group. A similar reduction in PEtOH levels was observed with application of either the PLD-linked mGluR antagonist, PCCG-13 (62.9±10.6, n = 7, ## p <0.01) or the mGluR1 antagonist, LY367385 (75.0±13.9, n = 12, ## p <0.01), while the mGluR5 antagonist, MPEP, did not decrease basal PLD activity (305.7±31.5, n = 7, ns) within the cocaine CPP group but were significantly increased compared to (* p <0.05) the saline treated group. Applications of SKF81297 (184.9±30.5, n = 12), SCH23390 (84.9±38.9, n = 12), MPEP (74.2±16.3, n = 7), LY367385 (94.7±18.9, n = 12) and PCCG-13 (132.5±18.4, n = 7) did not significantly alter the PEtOH levels in the saline-treated group compared to the basal activity levels. Inset is a depiction of the triangular excision performed to isolate amygdala (bilaterally for each animal, each slice) containing the basolateral (BLA), the central (CeA) and the lateral (LA) subregions from three serial coronal slices (350 µm) beginning −2.30 mm to −2.80 mm from Bregma .

Article Snippet: Primary antibodies (with the references provided where the antibodies were tested for specificity) included: metabotropic glutamate receptors [mGluR1 (AGC-006) and mGluR5 (AGC-007) ] from Alomone (Jerusalem, Israel); phospholipase D [PLD1 (sc-25512) and PLD2 (sc-25513), ] and actin (sc-1616) from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA); dopamine receptor D1R (AB20066) from Abcam (Cambridge, MA); D5R (MAB5292) from Millipore (Temecula, CA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clone 6C5) from Advanced Immunochemicals Inc. (Long Beach, CA).

Techniques: Activity Assay