antagonist fk888  (Tocris)

Journal: bioRxiv

Article Title: Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

doi: 10.1101/2023.07.04.547596

Figure Lengend Snippet: (A) Protocol for treatment of WT mice with either 3 i.n. doses every other day of PBS (gray), FAM with blank peptide conjugated to saporin (FAM 3X+Blank-SP, blue), [Sar ,Met(O 2 ) ]–Substance P conjugated to saporin (SSP-SAP 3X, green), or FAM with SSP-SAP (SSP-SAP 3X + FAM 3X, red) analyzed on D5. Data depicted in (C), (E), (G), (H), (K), (L), (O), and (P). (B) Protocol for treatment of WT mice with 3 i.n. doses every other day of either the NK1R inhibitor FK888 and PBS (FK888 3X + PBS 3X, gray), vehicle with FAM (Vehicle 3X + FAM 3X, blue), the NK1R inhibitor with Sar-SP (FK888 3X + Sar-SP 3X, green), or FK888 with FAM (FK888 3X + FAM 3X, red). Data depicted in (D), (F), (I), (J), (M), (N), (Q), and (R). (C-D) Sinonasal wash levels of IL-5 (left) and IL-13 (right) in (C) protocol (A), ablate or (D) protocol (B), inhibit. (E-F) Sinonasal wash levels of (E) IL-25 (left) and CysLT (right) for protocol (A, ablate) or (F) for protocol (B, inhibit). (G-J) Total number of respiratory tissue (G,I) Tuft-1 and (H,J) Tuft-2 cells in the (G-H) protocol (A), ablate or (I-J) in protocol (B), inhibit. (K-N) Total number of respiratory tissue (K, M) Ki-67+ basal cells and (L, M) ILC2s in (K-L) protocol (A), ablate or (M-N) protocol (B) inhibit. (O-R) Total number of respiratory tissue (O, Q) CD4+ TH2 cells and (P, R) eosinophils in (O-P) protocol (A), ablate or (Q-R) protocol (B), inhibit. Data are mean ± SEM, representative of 2 experiments (4-5 mice/treatment), one-way ANOVA with Tukey’s post-hoc tests. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also and .

Article Snippet: To inhibit NK1R, mice received 30 nmol of the antagonist FK888 (Tocris, cat #2400), which has been previously used i.n. in guinea pigs .

Techniques:

Journal: bioRxiv

Article Title: Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

doi: 10.1101/2023.07.04.547596

Figure Lengend Snippet: (A-K) Outcomes in ablation experiment following PBS (gray), FAM 3X + Blank-SAP (blue), SSP-SAP 3X (green), or SSP-SAP 3X + FAM 3X (red) for: (A) sneezing-like behavior evoked by capsaicin (12µM i.n.), which was counted from 10 min sound and video recording in a treatment-blind manner, numbers and/or percentages of all (B) Tuft-1 cells, (C) NK1R+ Tuft-1 cells, (D) all Tuft-2 cells, (E) NK1R+ Tuft-2 cells, (F) total basal cells, (G) Ki67+ basal cells, (H) NK1R+ basal cells, (I) ILC2, (J) CD4+ TH2 cells, and (K) eosinophils in the respiratory epithelium assessed by flow cytometry. (L-V) Outcomes of inhibitor experiment following PBS (gray), vehicle + FAM 3X (blue), FK888 3X +SAR-SP 3X (green), or FK888 3X + FAM 3X (red) for: (L) sneezing-like behavior evoked by capsaicin, numbers and/or percentages of all (M) Tuft-1 cells, (N) NK1R+ Tuft-1 cells, (O) all Tuft-2 cells, (P) NK1R+ Tuft-2 cells, (Q) total basal cells, (R) Ki67+ basal cells, (S) NK1R+ basal cells, (T) ILC2, (U) CD4+ TH2 cells, and (V) eosinophils in the respiratory epithelium assessed by flow cytometry. Data are mean ± SEM, representative of 2 experiments (4-8 mice/treatment) one-way ANOVA with Tukey’s post-hoc tests. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: To inhibit NK1R, mice received 30 nmol of the antagonist FK888 (Tocris, cat #2400), which has been previously used i.n. in guinea pigs .

Techniques: Flow Cytometry

Journal: Frontiers in Cellular Neuroscience

Article Title: Gap Junctions Between Striatal D1 Neurons and Cholinergic Interneurons

doi: 10.3389/fncel.2021.674399

Figure Lengend Snippet: Tetanic photostimulation of D1 neurons induces an inward current in ChI that could not be inhibited by SP blockers. (A) Schematic representation showing the injection of AAV2/2-retro-hSyn-Flpo into the substantia nigra pars reticulata (SNR) and of AAV-fDIO-ChR2-EYFP and AAV-DIO-mCherry into the striatum of a ChAT-Cre mouse. (B) Images showing the expression of virus in the striatum (left panel), scale bar = 500 μm. The zoom-in view of the dashed rectangular area, showing the specific expression of ChR2 in D1 neurons and the specific expression of mCherry in ChIs (right panel). Green: D1 neurons with ChR2-EYFP; magenta: ChIs with mCherry. Scale bars = 50 μm. (C) A schematic diagram showing the method of whole-cell recording of the ChR2-expressing D1 neuron by 470 nm stimulation. Trains of brief laser pulse at 5, 10, 20, and 50 Hz (5 ms duration, 5 mW) produced precise firing of action potentials ( n = 7 cells tested). (D) A schematic diagram and example traces (left) show brief optogenetic stimulation of ChR2-expressing D1 neurons produced a fast IPSC in ChI and the IPSC was inhibited by picrotoxin. The right panel shows group data. * p < 0.05 (paired t -test; n = 7 cells; ctrl: 231.6 ± 68.6 pA; mean ± SEM; picrotoxin: 25.2 ± 11.0 pA; recorded at −10 mV). (E) Images showing the specific expression of NK1R and ChAT in the striatum. White solid arrowheads indicated colocalized neurons. Scale bar = 100 μm. (F) A schematic diagram and example traces show that puffing 1 μM [Sar 9 , Met(O 2 ) 11 ]-substance P evoked inward currents from ChIs ( n = 9 cells; peak amplitude: −75.1 ± 14.5 pA). (G) The group data show the effect of the SP blockers cocktail on the response of ChIs to the puffing of 5 μM substance P. * p < 0.05 (paired t -test; n = 7 cells; ctrl: −118.7 ± 31.5 pA; SP blockers: −41.4 ± 11.4 pA). (H) Example traces (left and middle) show the effects of picrotoxin and the SP blockers cocktail of L703606, FK888, and SR14033 on (left panel) the currents evoked by tetanic stimulation (20 Hz, 5 ms pulses, 5 s duration) of D1 neurons and recorded in a ChI. Summary data (right panel) show that the effects of SP blockers lack statistical significance. n.s., non-significant, p > 0.05 (paired t -test; n = 12 cells; ctrl: −102.5 ± 12.7 pA; SP blockers: −97.0 ± 18.6 pA at −65 mV). (I) Images show the expression of NK1R and SST in the striatum. White solid arrowheads indicated colocalized neurons. Scale bar = 200 μm. (J) A schematic diagram and example trace show the response of an SST neuron to the puffing of 1 μM [Sar 9 , Met(O 2 ) 11 ]-substance P ( n = 5 cells; peak amplitude: −12.5 ± 3.2 pA). (K) A schematic diagram and example trace show the lack of response of an SST neuron in response to 5 s continuous optogenetic stimulation of D1 cells in TTX solution ( n = 5 cells; peak amplitude: 4.8 ± 5.1 pA at −65 mV).

Article Snippet: Drugs used in the slice recordings were as follows: tetrodotoxin (TTX, 1 μM, Tocris Bioscience, Bristol, United Kingdom), a voltage-gated sodium channel blocker; picrotoxin (50 μM, Sigma-Aldrich, St. Louis, MO, United States), a blocker of GABA A receptors; 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μM, Sigma-Aldrich, St. Louis, MO, United States), an AMPA-type glutamate receptor blocker; 2-amino-5-phosphonovalerate (APV, 50 μM, Sigma-Aldrich, St. Louis, MO, United States), an NMDA-type glutamate receptor blocker; SR140333 (10 μM, Tocris Bioscience, Bristol, United Kingdom); FK888 hydrate (10 μM, Sigma-Aldrich, St. Louis, MO, United States) and L-703606 oxalate salt hydrate (100 nM, Sigma-Aldrich, St. Louis, MO, United States), NK1R blockers; carbenoxolone disodium (CBX, 200 μM, Tocris Bioscience, Bristol, United Kingdom) and quinine (200 μM, Sigma-Aldrich, St. Louis, MO, United States), gap junctions blockers; [Sar 9 , Met(O 2 ) 11 ]-substance P (1 μM, Tocris Bioscience, Bristol, United Kingdom), a selective NK1R agonist; and substance P (5 μM, Tocris Bioscience, Bristol, United Kingdom).

Techniques: Injection, Expressing, Produced