In a subset of experiments, the AMPA and NMDA receptor antagonists, 100 μM NBQX (Abcam) (Guo et al., 2016 (link)) and 250 μM D-APV (Tocris Bioscience) (Zhang et al., 2017 (link)), were injected in the fastigial nucleus near the recording electrodes. APV and NBQX were added to a Krebs solution with the following composition (in mM): 120 NaCl, 2 KCl, 1.2 MgSO4, 26 NaHCO3, 1.2 KH2PO4, 2 CaCl2, and 11 glucose, equilibrated with 95% O2–5% CO2 (pH 7.4). The solution containing the drugs was pre-loaded in a pneumatic picopump (PV820, World Precision Instruments), operated through adjustable air pressure, terminating in a 35G needle, positioned using a Patch-Star micromanipulator (Scientifica, Ltd.). After 15 min of single-unit recording, the solution was injected at the rate of 1 μl/5 min. It should be noted that the injection of GABAergic antagonists in the fastigial nucleus would not help discerning the origin of the pause, as it would affect the synapses coming from both local interneurons and PCs.
Pv820
Manufactured by World Precision Instruments
Sourced in United States
The PV820 is a precision pressure sensor designed for laboratory applications. It features a durable stainless steel construction and is capable of measuring pressures in a range of 0 to 820 psi. The device provides accurate and reliable pressure readings, making it suitable for a variety of experimental and research purposes.
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Lab products found in correlation
Cfse solution, by Thermo Fisher Scientific (2 mentions)
405 v1 thermal anemometer, by Testo (2 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (2 mentions)
D apv, by Bio-Techne (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Bapta, by Thermo Fisher Scientific (1 mentions)
Transferman nk2 micromanipulator, by Eppendorf (1 mentions)
Chlorpromazine hydrochloride, by Merck Group (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Dynasore, by Bio-Techne (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Mm 33, by World Precision Instruments (1 mentions)
Tricaine methanesulfonate, by Merck Group (1 mentions)
Alexa fluor 568 hydrazide, by Thermo Fisher Scientific (1 mentions)
P 1000, by Sutter Instruments (1 mentions)
Fluoview fvmpe rs two photon laser scanning microscope, by Olympus (1 mentions)
Maitai hp ds ol, by Spectra-Physics (1 mentions)
Insight ds ol, by Spectra-Physics (1 mentions)
Smz 168, by Motic (1 mentions)
P30 vertical micropipette puller, by Sutter Instruments (1 mentions)
26 protocols using pv820
1
Targeted Fastigial Nucleus Modulation via AMPA and NMDA Antagonists
2
Microinjection of Embryos for Imaging
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Embryos were glued onto a coverslip (see Time-lapse imaging), dehydrated for 10–15 min, and covered with 1:1 halocarbon oil 27:700. Pharmacological inhibitors were injected ventrally into the perivitelline space of stage 14–15 embryos using a Transferman NK2 micromanipulator (Eppendorf) and a microinjector (PV820; World Precision Instruments) coupled to our spinning disk confocal microscope. Drugs were injected immediately before wounding and imaging. Drug solutions are predicted to be diluted 50-fold in the embryo (Foe and Alberts, 1983 (link)). Dynasore (Tocris Bioscience) was injected at 50 mM in 50% DMSO, chlorpromazine hydrochloride (Sigma-Aldrich) was injected at 50 mM in water, BAPTA (Life Technologies) was injected at 50 mM in water, thapsigargin (Sigma-Aldrich) was injected at 500 µM in 50% DMSO, and Y-27632 (Tocris Bioscience) was injected at 10 mM in water. 50% DMSO and water were used as controls.
3
Zebrafish Infection Model for Evaluating Antibacterial Treatments
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Prior to infection, embryos were manually dechorionated at 24-hpf stage and kept at 28°C. Aiming to establish a fast systemic infection (Milivojevic et al., 2018 (link)), 24-hpf dechorionated embryos were anesthetized with 150 μg mL–1 of tricaine (MS-222) solution and microinjected with ∼5 nL containing 600–700 P. aeruginosa PAO1-GFP cells into the circulation valley by a pneumatic picopump (PV820, World Precision Instruments, Sarasota, FL, United States). The number of injected viable bacteria was determined by counting bacteria from a drop cultured on LB agar plates at 37°C for 1 day. Injected embryos were allowed to recover for 1.5 h at 28°C, dead embryos were discarded, and then live embryos were transferred to 24-well plates containing 1 mL of E3 medium and 10 embryos per well. The infected embryos were treated with the different concentrations of YtnP-ZP1, antibiotics, and their combinations, and maintained at 30°C by 120 hpf. The embryos injected with 2% PVP were used as the control groups. Twenty embryos were used per concentration, and the experiment was performed twice. The survival and development of P. aeruginosa PAO1-infected embryos were recorded daily until 120 hpf (corresponding to fourth day post-infection). Antibacterial efficacy of applied treatments was determined according to the survival rate of treated embryos in relation to those in the untreated group.
4
Chick Embryo Cell Grafting Protocol
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Embryonated eggs were incubated at 38.5°C in a humidified incubator until HH14 stage. Cell lines or patient samples were dissociated and labeled with 8 μM CFSE solution (Life Technologies). Stage HH14 chick embryos were grafted with fluorescent cells in presumptive somitic areas or in the brain parenchyma, with a glass capillary connected to a pneumatic PicoPump (PV820, World Precision Instruments) under a fluorescence stereomicroscope. Targeted tissue areas for the graft were visualized under the stereomicroscope. For cell lines, approximately 2500 living cells were grafted in each embryo, 200 to 300 for patient samples. For patient samples, the full cellular content obtained after dissociation was engrafted including stromal and/or immune cells.
5
Imaging Transgenic Zebrafish Larvae
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All imaging experiments were done on 3 dpf Tg(sspo:sspo-GFP), Tg(sspo:sspo-GFP;pkd2l1:tagRFP;pkd2l1:GCaMP5G), and Tg(sspo:sspo-GFP;β-actin:Arl13b-GFP) zebrafish larvae. Their respective siblings were used as controls (i.e., wild-type and heterozygous mutants from the same clutch). For all experiments, larvae were laterally mounted in glass-bottom dishes (MatTek, Ashland, Massachusetts, USA), filled with 1.5% low-melting point agarose. Unless otherwise noted, larvae were paralyzed by injecting 1–2 nL of 500 mM alpha-bungarotoxin (TOCRIS) in the caudal muscles of the trunk via glass micropipette held by a micromanipulator (Märzhäuser Wetzlar MM-33), using a pneumatic Pico-pump (World Precision Instruments PV-820).
6
Tracking Vitamin B12 Derivatives in Zebrafish
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To determine the distribution and the accumulation of B12-JR1 and B12-JR1-CBC through inner organs, transgenic Tg(fabp:EGFP) zebrafish embryos were exposed to a non-toxic concentration of each of the tested compounds (150 µM of B12-JR1 and 20 µM B12-JR1-CBC ) in a period from 72 to 120 hpf (a long exposure started when the liver became vascularized and capable to metabolize the absorbed compounds) and 106 to 120 hpf (a shorter exposure time). To verify a preferential accumulation of B12-JR1-CBC in the liver, the compound was applied intravenously (parenteral use) to zebrafish embryos. The 106-hpf old embryos were anesthetized by addition of tricaine-methane sulfonate (200 μg/mL, Sigma-Aldrich), and microinjected by a pneumatic picopump (PV820, World Precision Instruments, USA) with 5 nL of B12-JR1-CBC (9.56 µg/nL per an embryo, corresponding to 20 µM dose applied into embryo water). The treated embryos were analysed at 120 hpf by a fluorescent microscope (Olympus BX51, Applied Imaging Corp., San Jose, CA, USA) upon Texas Red filter (an excitation max 596 nm, an emission max 620 nm) and Spectrum Green filter (an excitation max 497 nm, an emission max 524 nm) to detect B12-JR1-CBC and the EGFP-labelled liver, respectively.
7
Cricket Behavior Analysis via Air-Puff and Acoustic Stimuli
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An air-puff stimulus was provided to the stationary cricket by a short puff of nitrogen gas from a plastic nozzle (ø = 15 mm) connected to a pneumatic picopump (PV820, World Precision Instruments, Sarasota FL, USA). For the behavioral experiments using the treadmill system, two air-puff nozzles were arranged in parallel to align their center to the same horizontal plane as the top of the treadmill sphere (Figure 1A ). Nozzle ends were placed at 200 mm from the animal. The velocity of the air puffs was controlled at 1.00 m/s, which was measured at the center of the arena with a 405-V1 thermal anemometer (Testo, Yokohama, Japan), by adjusting the delivery pressure of the picopump. The duration of the air-puff stimulus was 200 ms. By changing the cricket’s body axis relative to the stimulus nozzle, the air puffs were applied to the right or left side of the cerci from one of the air nozzles, which was close to the posterior side of the crickets.
The acoustic stimuli were 15-kHz pure tones, synthesized using RPvdsEx software (Tacker Davis Technologies, Alachua FL, USA), and transduced and attenuated using an RM1 processor (TDT). The sounds were calibrated at an average of 70 dB SPL and delivered by a 1.5-inch (3.81 cm) full-range sealed loudspeaker (MM-SPS2, Sanwa Supply, Okayama, Japan), which was located just above the air-puff nozzles at 200 mm away from the animal (Figure 1A ).
The acoustic stimuli were 15-kHz pure tones, synthesized using RPvdsEx software (Tacker Davis Technologies, Alachua FL, USA), and transduced and attenuated using an RM1 processor (TDT). The sounds were calibrated at an average of 70 dB SPL and delivered by a 1.5-inch (3.81 cm) full-range sealed loudspeaker (MM-SPS2, Sanwa Supply, Okayama, Japan), which was located just above the air-puff nozzles at 200 mm away from the animal (
8
Generation of Transgenic Zebrafish Expressing EGFP under col2a1a Promoter
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The plasmid col2a1a:EGFP was constructed using the Tol2/Gateway kit (Kwan et al., 2007 (link)). The col2a1a promoter was amplified using gDNA extracted from embryos as a template and cloned into the p5ʹE entry vector. The EGFP and polyA tails were cloned into the pME and p3ʹE entry vectors, respectively. These were then assembled into the Tol2 destination vector using the MultiSite Gateway Technology system (Invitrogen). One-cell stage embryos were microinjected with 50 ng/μL Tol2 mRNA and 50 ng/μL of col2a1a:EGFP construct using a pneumatic pico-pump (PV-820, World Precision Instrument). F0 zebrafish were crossed with wild-type AB to generate F1 embryos, which were screened for GFP expression. F0 zebrafish that could produce germ-line GFP expression were crossed to produce F1 heterozygotes. F2 generations were generated by F1 heterozygotes in-cross.
9
Antioxidant and Anti-inflammatory Effects of HDL Subclasses
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To compare antioxidant and anti-inflammatory activities of HDL between slim and plump, HDL3 from each group was injected into zebrafish embryos, as in our previous report (32 (link)). Embryos at 1-day post-fertilization (dpf) were individually microinjected using a pneumatic picopump (PV820; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) and a pulled microcapillary pipette-using device (PC-10; Narishigen, Tokyo, Japan). To minimize bias, injections were performed at the same position on each yolk. Filter-sterilized solution of each LDL or HDL3 was injected into flasks of embryos. Following injection, live embryos were observed under a stereomicroscope (Motic SMZ 168; Hong Kong) and photographed using a Motic cam2300 CCD camera.
10
CRISPR Microinjection for Embryo Editing
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