Journal: bioRxiv

Article Title: A GRAB sensor reveals activity-dependent non-vesicular somatodendritic adenosine release

doi: 10.1101/2020.05.04.075564

Figure Lengend Snippet: (A) Blocking synaptic vesicle release with tetanus neurotoxin (TeNT) abolishes glutamate release, but not Ado release. (A1) Schematic drawing depicting the release of neurotransmitter-containing synaptic vesicles, which is blocked by TeNT. (A2) Representative confocal micrographs showing hippocampal neurons expressing TeNT (blue), Ado1.0 (green), and R ncp -iGlu (red). (A3 and A4) Averaged traces (A3) and group summary (A4) of Ado1.0 (upper panels, green) and R ncp -iGlu (bottom panels, red) ΔF/F 0 in response to field stimuli (30 Hz, 100 pulses) with or without TeNT expression (n = 4 coverslips per group); the data in the control group are reproduced from . (B) Blocking CD39 or knocking out CD73 does not affect activity-dependent Ado release. (B1) Schematic drawing depicting the production of Ado from ATP via the CD39 and CD73 enzymes. (B2 and B3) Averaged traces (B2) and group summary (B3) of Ado1.0 ΔF/F 0 in response to field stimuli (30 Hz, 100 pulses) under control conditions, in the presence of the CD39 inhibitor POM1 (10 μM), and in CD73 knockout neurons; n = 4-5 coverslips per group. The inset in (B2) shows example PCR genotyping results of CD73 KO mice. (C) Inhibiting ENT blocks activity-dependent Ado release, but not glutamate release. (C1) Schematic drawing depicting the release of Ado via equilibrative nucleoside transporters (ENTs). (C2 and C3) Averaged traces (C2) and group summary (C3) of Ado1.0 (upper panels, green) and R ncp -iGlu (bottom panels, red) ΔF/F 0 in response to high K + before (control), during, and after (wash) application of NBTI (5 μM) and dipyridamole (DIPY, 10 μM); n = 7 coverslips each.

Article Snippet: Solutions containing adenosine (Sigma), ADP (Sigma), ATP (Sigma), SCH-58261 (Abcam), HENECA (Tocris), inosine (Sigma), adenine (Sigma), CdCl 2 (Sigma), L-glutamate (Sigma), bradykinin (Sangon Biotech Shanghai), thrombin (Sigma), POM1 (Santa Cruz), S-(4-nitrobenzyl)-6-thioinosine (NBTI, Santa Cruz), dipyridamole (Santa Cruz), ω-Conotoxin-GVIA (Tocris), ω-Agatoxin IVA (Cayman) nimodipine (Cayman), and (±)-felodipine (Cayman) were delivered via a custom-made perfusion system or via bath application.

Techniques: Blocking Assay, Expressing, Control, Activity Assay, Knock-Out

Journal: bioRxiv

Article Title: A GRAB sensor reveals activity-dependent non-vesicular somatodendritic adenosine release

doi: 10.1101/2020.05.04.075564

Figure Lengend Snippet: (A) Schematic drawing depicting the putative mechanism by which equilibrative nucleoside transporters (ENTs) mediate Ado release in neurons. (B and C) Averaged traces (B) and group summary (C) of Ado1.0 ΔF/F 0 in response to field stimuli (30 Hz, 100 pulses, indicated by black tick marks) before (control), during, and after (wash) application of the ENT1 blocker NBTI (5 μM) and/or the ENT1/2 blocker dipyridamole (DIPY, 10 μM); n = 4 coverslips.

Article Snippet: Solutions containing adenosine (Sigma), ADP (Sigma), ATP (Sigma), SCH-58261 (Abcam), HENECA (Tocris), inosine (Sigma), adenine (Sigma), CdCl 2 (Sigma), L-glutamate (Sigma), bradykinin (Sangon Biotech Shanghai), thrombin (Sigma), POM1 (Santa Cruz), S-(4-nitrobenzyl)-6-thioinosine (NBTI, Santa Cruz), dipyridamole (Santa Cruz), ω-Conotoxin-GVIA (Tocris), ω-Agatoxin IVA (Cayman) nimodipine (Cayman), and (±)-felodipine (Cayman) were delivered via a custom-made perfusion system or via bath application.

Techniques: Control

Journal: bioRxiv

Article Title: A GRAB sensor reveals activity-dependent non-vesicular somatodendritic adenosine release

doi: 10.1101/2020.05.04.075564

Figure Lengend Snippet: (A1) Left panel: sparse expression of Ado1.0 in cultured hippocampal pyramidal neurons. Right panel: fluorescence images and pseudocolor images of Ado1.0 ΔF/F 0 in the somatodendritic (purple box) and axonal (blue box) compartments in response to field stimuli (30 Hz, 100 pulses), high K + , or Ado (100 nM); scale bar, 50 μm. Example traces and summary data are shown in (A2) and (A3) , respectively; n = 11-21 ROIs from 4 coverslips per group. (B) Schematic illustration depicting the strategy used to image acute hippocampal brain slices prepared from mice expressing Ado1.0med and tdTomato-ChrimsonR in the CA1 region while using a 633-nm laser to activate the neurons. At the right is an image showing Ado1.0med fluorescence; the box shows the region magnified in (C) . (C) Magnified fluorescence images of the CA1 region showing Ado1.0med (green) and tdTomato-ChrimsonR (red) in CA1 regions (scale bar, 100 μm). The pyramidal cell layer (Str. py) is located between the stratum oriens (Str. ori) and stratum radiatum (Str. rad). (D) Pseudocolor images (left), averaged traces (middle), and group summary (right) of Ado1.0med ΔF/F 0 in response to 633-nm laser pulses applied at 20 Hz for the indicated duration (scale bar, 100 μm); the solid line shown in the right panel a linear fit to the data; n = 6 slices from 4 mice. (E) Blocking L-type VGCCs inhibits optogenetically induced Ado release. Where indicated, nimodipine (Nim, 20 μM) and Cd 2+ (100 μM) were applied (scale bar, 100 μm); n = 5 slices from 3 mice. (F) Blocking ENT transporters inhibits optogenetically induced Ado release. Where indicated, dipyridamole (DIPY, 20 μM) was used to block ENTs. Averaged traces (F1) and the group summary (F2) of Ado1.0med ΔF/F 0 are shown; n = 4 slices from 3 mice. (G) Model depicting the novel mode of neuronal activity–dependent Ado release from hippocampal neurons. Ado is released slowly from the postsynaptic membrane via a vesicle-independent, ENT-dependent mechanism, serving as a putative retrograde signal to regulate presynaptic activity. This activity-dependent release of Ado requires L-type voltage-gated Ca 2+ channels (VGCCs). In contrast, classic neurotransmitters such as glutamate (Glu) are released from presynaptic vesicles requires P/Q-type and N-type VGCCs. AdoR, adenosine receptor; ENTs, equilibrative nucleoside transporters; Syts, synaptotagmins.

Article Snippet: Solutions containing adenosine (Sigma), ADP (Sigma), ATP (Sigma), SCH-58261 (Abcam), HENECA (Tocris), inosine (Sigma), adenine (Sigma), CdCl 2 (Sigma), L-glutamate (Sigma), bradykinin (Sangon Biotech Shanghai), thrombin (Sigma), POM1 (Santa Cruz), S-(4-nitrobenzyl)-6-thioinosine (NBTI, Santa Cruz), dipyridamole (Santa Cruz), ω-Conotoxin-GVIA (Tocris), ω-Agatoxin IVA (Cayman) nimodipine (Cayman), and (±)-felodipine (Cayman) were delivered via a custom-made perfusion system or via bath application.

Techniques: Expressing, Cell Culture, Fluorescence, Blocking Assay, Activity Assay, Membrane

Journal: Pharmaceutical Research

Article Title: CLSM as Quantitative Method to Determine the Size of Drug Crystals in a Solid Dispersion

doi: 10.1007/s11095-011-0484-8

Figure Lengend Snippet: Composition of the Different Solutions Used to Prepare the Controlled Crystallized Dispersions. In All Experiments, 1.2 mL Aquous Solution was Mixed With 0.8 mL TBA Solution (i.e. ratio of 6/4)

Article Snippet: Dipyridamole and TBA were obtained from Sigma-Aldrich Chemie B.V. Zwijndrecht, The Netherlands.

Techniques: Lyophilization

Journal: Pharmaceutical Research

Article Title: CLSM as Quantitative Method to Determine the Size of Drug Crystals in a Solid Dispersion

doi: 10.1007/s11095-011-0484-8

Figure Lengend Snippet: X-ray diffraction patterns of δ-mannitol, dipyridamole as received, and the dispersions that were prepared freeze-drying from solutions containing 25.0 and 50.0 mg/mL dipyridamole in TBA. In the patterns of the solid dispersions, the major dipyridamole peak at 8.1° 2θ is clearly visible, but also minor dipyridamole peaks at 8.9, 10.3, 18.8, 20.9, 23.5, and 26.1° 2θ were found. Both dispersions contained 10% w/w dipyridamole in mannitol.

Article Snippet: Dipyridamole and TBA were obtained from Sigma-Aldrich Chemie B.V. Zwijndrecht, The Netherlands.

Techniques:

Journal: Pharmaceutical Research

Article Title: CLSM as Quantitative Method to Determine the Size of Drug Crystals in a Solid Dispersion

doi: 10.1007/s11095-011-0484-8

Figure Lengend Snippet: SEM pictures of two controlled crystallized dispersions. Both contain 10% w/w dipyridamole in mannitol, but one was prepared from a solution containing 25.0 mg/mL dipyridamole in TBA ( top ), while the other was prepared from a solution containing 50.0 mg/mL dipyridamole in TBA ( bottom ). Both pictures have a magnification of 5000x.

Article Snippet: Dipyridamole and TBA were obtained from Sigma-Aldrich Chemie B.V. Zwijndrecht, The Netherlands.

Techniques:

Journal: Pharmaceutical Research

Article Title: CLSM as Quantitative Method to Determine the Size of Drug Crystals in a Solid Dispersion

doi: 10.1007/s11095-011-0484-8

Figure Lengend Snippet: Examples of a merged image of the brightfield reflection image and fluorescent dipyridamole images ( left ), fluorescent images ( middle ), and binary images ( right ) of the controlled crystallized dispersions obtained from the solution containing 25 mg/mL ( top ) and 50 mg/mL ( bottom ) dipyridamole in TBA. Both controlled crystallized dispersions contain 10% w/w dipyridamole in mannitol.

Article Snippet: Dipyridamole and TBA were obtained from Sigma-Aldrich Chemie B.V. Zwijndrecht, The Netherlands.

Techniques:

Journal: Pharmaceutical Research

Article Title: CLSM as Quantitative Method to Determine the Size of Drug Crystals in a Solid Dispersion

doi: 10.1007/s11095-011-0484-8

Figure Lengend Snippet: ( a ) Particle size distribution of the crystalline dispersions obtained from the 25 mg/mL ( light grey ) and 50 mg/mL ( dark grey ) dipyridamole in TBA solution and the physical mixture ( black ). ( b ) Dissolution profiles of dipyridamole from tablets composed of the physical mixture ( filled circle ) and controlled crystallized dispersions. The dispersions were prepared from a solution containing 25 mg/mL ( white circle ) and 50 mg/mL ( filled circle ) dipyridamole in TBA. The D 50 -values in the figure are the values as determined in ( a ). All tablets contained 10% w/w dipyridamole ( n = 3–6; mean ± standard deviation).

Article Snippet: Dipyridamole and TBA were obtained from Sigma-Aldrich Chemie B.V. Zwijndrecht, The Netherlands.

Techniques: Dissolution, Standard Deviation