PER2::LUC mice 4 weeks of age were used for luciferase assay. Mice were sacrificed, and dissected brains were immediately cooled in ice-cold Hank’s Balanced Sodium Saline. The piriform cortex and the SCN were further coronally dissected in 150μm-thickness with vibratome. Sectioned brain slices were placed on Milli Cell immersed in culture medium (5% MEM, 18% HBSS, 15 mM NaHCO3, 2 mM HEPES, 10 mM D-glucose, 1 mM L-glutamine, 175 mg/L Ascorbic Acid, 1 mg/mL insulin in 0.01 M HCl, 25% Horse Serum; pH adjusted to 7.0~7.4) with Luciferin, and incubated at 37 °C, 5% CO2. Luminescence was detected by Kronos Dio (Atto) with 1-min exposure, 10-min intervals. For tetrodotoxin application, tetrodotoxin (Fujifilm) was applied to the culture medium with a final concentration of 20 µM.
Tetrodotoxin
Manufactured by Fujifilm
Sourced in Japan
Tetrodotoxin is a potent neurotoxin found in various marine organisms, including pufferfish. It is commonly used in scientific research as a laboratory tool to study voltage-gated sodium channels and their role in nerve impulse transmission.
Automatically generated - may contain errors
Lab products found in correlation
Bicuculline methiodide, by Merck Group (2 mentions)
Pds1000 he biolistic gene gun, by Bio-Rad (2 mentions)
Vt1200, by Leica camera (2 mentions)
Millicell culture insert, by Merck Group (2 mentions)
Trolox, by Merck Group (2 mentions)
Lactacystin, by Merck Group (2 mentions)
Suramin, by Merck Group (2 mentions)
4 aminopyridine 4 ap, by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
R software environment, by R Project for Statistical Computing (1 mentions)
Picrotoxin, by Merck Group (1 mentions)
6 cyano 7 nitroquinoxaline 2 3 dione cnqx, by Merck Group (1 mentions)
Gsk1016790a, by Bio-Techne (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Tokyo Chemical Industry (1 mentions)
Acetylcholine, by Fujifilm (1 mentions)
L name, by Merck Group (1 mentions)
Carbachol, by Fujifilm (1 mentions)
Apamin, by Peptide Institute (1 mentions)
13 protocols using tetrodotoxin
1
Circadian Rhythms in Transgenic Mice
2
Evaluating Excitatory-Inhibitory Balance in Neurons
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For miniature recordings, we used a modified intracellular solution to adjust the reversal potential of the GABAA receptor response [127.5 mM cesium methanesulfonate, 7.5 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, 0.6 mM EGTA (pH 7.25)]. Moreover, we added 0.5 µM tetrodotoxin (Wako Pure Chemicals, Osaka, Japan) to perfusate to block action potentials. The voltage was clamped at −60 mV for mEPSC recording and at 0 mV for mIPSC recording (Figs. 2A and 3A ). We analyzed the frequency and amplitude of mEPSCs and mIPSCs above 10 pA.
We obtained 4 miniature parameters (mean mEPSC amplitude, mean mIPSC amplitude, mean mEPSC frequency, and mean mIPSC frequency) in individual CA1 pyramidal neurons. For graphic expression, the distribution was visualized 2-dimensionally in the R software environment (R Foundation for Statistical Computing, Vienna, Austria) (amplitude inFig. 2B Fig. 3B
We obtained 4 miniature parameters (mean mEPSC amplitude, mean mIPSC amplitude, mean mEPSC frequency, and mean mIPSC frequency) in individual CA1 pyramidal neurons. For graphic expression, the distribution was visualized 2-dimensionally in the R software environment (R Foundation for Statistical Computing, Vienna, Austria) (amplitude in
3
Biolistic Transfection of Hippocampal Slices
4
Electrophysiological Characterization of Synaptic Responses
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All electrophysiological experiments were performed using the patch-clamp method, where the membrane potential was clamped at −70 mV. Evoked EPSCs and depolarization-induced Na+ currents were recorded in response to an action potential elicited by a brief (2 ms) somatic depolarization pulse (to 0 mV) from the patch pipette under voltage-clamp conditions. Spontaneous mEPSCs were recorded in the presence of an Na+ channel inhibitor, 0.5 μM tetrodotoxin (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), in the extracellular fluid. Synaptic responses were recorded at a sampling rate of 20 kHz and were filtered at 10 kHz. Data were excluded from the analysis if a leak current >300 pA was observed. The data were analyzed offline using AxoGraph X 1.2 software (AxoGraph Scientific).
Extracellular fluid (pH 7.4) was as follows (mM): NaCl 140, KCl 2.4, HEPES 10, glucose 10, CaCl2 2, and MgCl2 1. Some experiments were performed under Mg2+-free/10 μM glycine conditions. In these cases, the composition of the extracellular solution was modified only with regard to Mg2+ and glycine without readjusting the concentration of the other ions. Intracellular fluid (pH 7.4) was as follows (mM): K-gluconate 146.3, MgCl2 0.6, ATP-Na2 4, GTP-Na2 0.3, creatine phosphokinase 50 U/mL, phosphocreatine 12, EGTA 1, and HEPES 17.8.
Extracellular fluid (pH 7.4) was as follows (mM): NaCl 140, KCl 2.4, HEPES 10, glucose 10, CaCl2 2, and MgCl2 1. Some experiments were performed under Mg2+-free/10 μM glycine conditions. In these cases, the composition of the extracellular solution was modified only with regard to Mg2+ and glycine without readjusting the concentration of the other ions. Intracellular fluid (pH 7.4) was as follows (mM): K-gluconate 146.3, MgCl2 0.6, ATP-Na2 4, GTP-Na2 0.3, creatine phosphokinase 50 U/mL, phosphocreatine 12, EGTA 1, and HEPES 17.8.
5
Patch-Clamp Recording of Synaptic Currents
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Glass electrodes were pulled from thin-walled borosilicate glass capillaries using a micropipette puller (Model P-1000IVF, Sutter Instrument, Novato, CA, USA). To record the synaptic currents, we used recording glass electrodes with 3- to 7-megohm tip resistance when filled with an internal solution [65 mM K-gluconate, 70 mM KCl, 10 mM Hepes, 0.5 mM EGTA, 1 mM MgCl2, 12 mM Na-phosphocreatine, 4 mM Mg–adenosine triphosphate (ATP), 0.5 mM Na–guanosine triphosphate (GTP), and 0.2% biocytin, adjusted to pH 7.2 to 7.3 with KOH]. Voltage-clamp recordings were performed at a holding potential of −70 mV. To measure sIPSCs, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM; Sigma-Aldrich, St. Louis, MO, USA) and 2-amino-5-phosphonopetanoic-acid (AP5; 50 μM; Abcam) were added to ACSF to exclude EPSCs. To measure mIPSCs, CNQX (10 μM), AP5 (50 μM), and tetrodotoxin (1 μM; Wako, Osaka, Japan) were added to ACSF to exclude EPSCs and IPSCs caused by network activity. To measure sEPSCs, picrotoxin (100 μM; Sigma-Aldrich) was added to ACSF to exclude IPSCs via GABAA (γ-aminobutyric acid type A) receptors. Synaptic currents were recorded for 1 min, and 20 recordings from each cell were used to determine the cumulative probability.
6
Preparation of Stock Solutions for Cell Experiments
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Stock solutions were prepared by dissolving 9-phenanthrol (9-phe, Tocris) and GSK 1016790A (GSK, Tocris) in DMSO. Cyclic ADP ribose was freshly dissolved on each experiment day. Tetrodotoxin (Wako) was dissolved in distilled water to prepare stock solutions. LPS were purchased from Sigma-Aldrich. The dissolved chemicals were diluted 1:1,000 in the medium and solution for time-lapse imaging and patch-clamp experiments, respectively. The final DMSO concentration did not exceed 0.1%.
7
Krebs's Solution for Tissue Maintenance
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During the experiments, tissues were maintained in Krebs’s solution consisting of (mM): NaCl 118.4, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11.7. ATP was obtained from Tokyo Chemical Industry (Tokyo, Japan). Tetrodotoxin was obtained from FUJIFILM Wako (Osaka, Japan). Suramin and CBF3GA, which were used as antagonists for purinergic receptors [37 (link),38 (link)], were obtained from Sigma-Aldrich (St Louis, MO, USA). The drugs were dissolved in distilled water. We confirmed that the highest concentration of vehicles (0.1%) for the drugs alone had no effect on the basal tone and contractile responses at the concentrations used. Final concentrations in the bath solution were described as the concentrations of drugs.
8
Examining Gastrointestinal Motility via Erythromycin
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Human motilin was purchased from Anygen Co., Ltd (GwangJu, Korea). Erythromycin was purchased from Mylan (Tokyo, Japan). Acetylcholine (ACh), atropine and tetrodotoxin (TTX) were purchased from Wako Pure Chemical (Osaka, Japan). For in vivo experiments (gastric emptying), erythromycin was diluted in physiological salt solution. The dose of erythromycin was expressed as mg/kg body weight. For in vitro experiments (contraction), erythromycin was diluted in, and other drugs were dissolved in, physiological salt solution. Drug concentrations were expressed as final molar concentrations in the organ bath solution. All drugs were prepared immediately before use.
9
Biolistic Transfection of Hippocampal Slices
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Hippocampal slices (350-μm thick) were dissected from SD rats at P7 by a vibratome (VT1200S; Leica, Wetzlar, Germany), mounted onto 0.4-μm Millicell™ culture inserts (EMD Millipore, Billerica, MA). At DIV 11, slices were transfected biolistically by a PDS1000/He Biolistic Gene Gun (Bio-Rad, Hercules, CA) with 1.6-μm gold microcarriers. At 2- to 4-days after transfection, cultures were transferred to the recording chambers and constantly perfused with oxygenated artificial cerebrospinal fluid (ACSF, 95% O2 and 5% CO2) containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 20 mM glucose, and 200 μM Trolox (Sigma-Aldrich, St. Louis, MO) at 29–30°C. In some experiments, we added tetrodotoxin (Wako, Osaka, Japan, 1 μM), bicuculline methiodide (Sigma-Aldrich, 12 μM), lactacystin (EMD Millipore, 10 μM) to culture and the recording medium.
10
Mechanical Response Experiments in Modified Tyrode's Solution
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During experiments for recording mechanical responses, tissues were maintained in modified Tyrode's solution consisting of (in mM) NaCl 136.9, KCl 2.68, CaCl2 1.80, MgCl2 1.0, NaH2PO4 0.41, NaHCO3 11.90, and glucose 5.55. tetrodotoxin was used as a blocker of voltage-dependent sodium channels on neurons. Atropine was used to block muscarinic acetylcholine receptors on smooth muscle cells. N-Acetyl-L-tryptophan 3,5-bis (trifluoromethyl) benzyl ester (L-732,138) and (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl] benzamide (SR48968) were used to block tachykinin NK1 and tachykinin NK2 receptors, respectively. Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) was used as a nitric oxide (NO) synthase (NOS) inhibitor. L-732,138 and L-NAME were obtained from Sigma (St. Louis, MO, USA). Atropine sulfate salt monohydrate, TNBS, and tetrodotoxin were obtained from Wako (Osaka, Japan). SR48968 was a gift from Sanofi-Synthelabo (Montpellier, France). L-732,138 and SR48968 were dissolved in ethyl acetate and ethanol, respectively. The vehicles (ethanol and ethyl acetate) for the drugs alone had no effect on basal tone. Other drugs were dissolved in distilled water. The drug concentrations given in the test were final concentrations in the bath solution.
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