Matrigel basement membrane matrix was purchased at Corning Life Sciences (354230). Matrigel protein concentration as obtained by the Lowry method ranged between 9.2 and 10.4 mg/ml and endotoxin as measured by Limulus amoebocyte lysate assay was <1.5 EU/ml. Collagenase, recombinant BDNF and insulin were purchased from Sigma (C9263, B3795 and I1882, respectively). Dispase II was purchased from Roche (Neutral protease, grade II, 04942078001). Chicken embryo extract was purchased at MP Biochemicals (MP Biochemicals, 2850145). Recombinant rat CNTF and recombinant rat GDNF were purchased at R&D Systems (557-NT-010 and 512-GF-010, respectively). Tetrodotoxin (TTX) was purchased at Alomone Labs (T-550). Tubocurarine was purchased at Sigma-Aldrich (T2379).
Tetrodotoxin ttx
Manufactured by Alomone
Sourced in Israel, Palestine, State of
Tetrodotoxin (TTX) is a highly potent neurotoxin isolated from various marine organisms, such as pufferfish. It is a sodium channel blocker that inhibits the flow of sodium ions across nerve cell membranes, thereby interrupting the transmission of electrical signals. TTX is commonly used in scientific research to investigate the role of sodium channels in neurophysiological processes.
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Lab products found in correlation
Nifedipine, by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Nicardipine, by Merck Group (2 mentions)
Ranolazine, by Merck Group (2 mentions)
Strychnine, by Merck Group (2 mentions)
4 aminopyridine 4 ap, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
6 cyano 7 nitroquinoxaline 2 3 dione cnqx, by Bio-Techne (2 mentions)
Dl 2 amino 5 phosphonovaleric acid ap5, by Bio-Techne (2 mentions)
Bicuculline methiodide, by Merck Group (2 mentions)
Dl ap5, by Bio-Techne (2 mentions)
Mouse kiss 1 110 119 nh2 kp 10, by Phoenix Pharmaceuticals (2 mentions)
Bicuculline methochloride, by Bio-Techne (2 mentions)
Mannitol, by Thermo Fisher Scientific (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Water immersion objective, by Olympus (2 mentions)
Glass capillary tube, by A-M Systems (2 mentions)
Pclamp 9, by Molecular Devices (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Tubocurarine, by Merck Group (1 mentions)
42 protocols using tetrodotoxin ttx
1
Isolation and Culture of Dorsal Root Ganglia
2
Tetrodotoxin (TTX) Protocol
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Tetrodotoxin (TTX) was purchased from Alomone Labs (Jerusalem, Israel). All other drugs were purchased from SIGMA. Drugs were prepared and stored at 1000× final concentration and diluted in bath perfusate prior to application.
3
Acute 5-HT Responses in L6 Neurons
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Acute responses to 5-HT were probed by bath application of 5-HT (serotonin creatinine sulfate, Sigma-Aldrich, St. Louis, MO; 10 µm ; 30 s) in ACSF. To examine the effect of 5-HT on the excitability of L6 pyramidal neurons, 5-HT (10 µm ) was bath applied until a steady-state response was reached, and remained in bath throughout the duration of the input–output test protocols (∼2 min total application). Selective antagonists and agonists were from Tocris (Bristol, UK), except where mentioned. Antagonists for 5-HT1A receptors (30 nm WAY100635, 10 µm NAN-190) and 5-HT2A receptors (30 nm MDL100907; 2 µm ketanserin; 300 nm to 3 µm ritanserin) were applied in bath for 10 min before further experiments with 5-HT. There were no significant differences between effects of 300 nm and 3 µm ritanserin, and results were grouped for analysis. TCB-2 was used as a specific agonist of 5-HT2A receptors (300 nm to 1 µm ). Other agonists and antagonists used for characterization of the 5-HT response in L6 neurons were as follows: 2 µm tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel), 20 µm 6-cyano-7-nitroquinoxaline-2,3-dione, 50 µm d -2-amino-5-phosphonovaleric acid, 100 µm picrotoxin, 1 µm CGP52432, and 10 µm 8-OH-DPAT.
4
Pharmacological Modulation of Neural Circuits
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In some experiments, 1 μM tetrodotoxin (TTX; Alomone Laboratories, Jerusalem, Israel) was administered to eliminate action potential generation in the preparation. The GluN2B-specific NMDA receptor blocker ifenprodil was used in 5 μM, whereas the general NMDA receptor antagonist D(-)-2-Amino-5-phosphonopentanoic acid (D-AP5) was administered in 50 μM concentration (Tocris Cookson Ltd., Bristol, UK). The glutamate transporter inhibitor DL-threo-β-Benzyloxyaspartic acid (DL-TBOA; Tocris Cookson Ltd., Bristol, UK) was applied in 10 μM concentration. In certain experiments, slices were treated with 1 μM thapsigargin for 45 min to inhibit astrocytic calcium wave activity (Kőszeghy et al., 2015 (link))., WIN55,212-2 (1 μM) was used as CB1 receptor agonist, and the muscarinic agonist carbamylcholine chloride (carbachol) was administered in 50 μM. Serotonin was administered in 10 μM. At the end of some experiments, slices were washed with 10 μM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), 50 μM D-AP5, 1 μM strychnine and 10 μM bicuculline (Tocris Cookson Ltd., Bristol, UK) in order to block fast synaptic neurotransmission.
5
Patch clamp recording of Nav1.6 and Nav1.8 channels
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We performed whole-cell patch clamp recordings with an extracellular solution consisting (mmol/l): 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 1 CaCl2 and 10 HEPES (or 10 MES fo acidic solutions). The pHvalue was adjusted the desired pH-values with HCl or TMA-OH. For recordings of Nav1.6 and Nav1.8, 300 nM tetrodotoxin (TTX, Alomone labs, Jerusalem, Israel) was included in all solutions in order to block all endogenous TTX-sensitive Na+ channels expressed in N1E115 cells. For the experiments we replaced the medium of the cell culture dishes with the extracellular solution (pH 7.4) and applied the test-solutions in increasing pH-values using a self-made, gravity driven application system. The pipette solution contained (mmol/l): 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES. The pH-value was adjusted to 7.4 with CsOH. All solutions were stored at 7 °C in light-excluding bottles and used within one month.
6
Pharmacological Modulation of Neuronal Activity
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Drugs were diluted in distilled water to make stock solutions and then again in saline to the working concentration on the day of experimentation. Drugs used were hyoscine (1 μM), nicardipine (1.25 μM) (both from Sigma Aldrich), CT (12.5 μg/ml, List Biologicals, Campbell, CA), tetrodotoxin (TTX, 1 μM, Alomone Labs, Jerusalem, Israel), granisetron (1 μM) (Smith Kline Beecham, Harlow Essex, UK), neurokinin 1 (NK1) antagonist SR140333 (100 nM) and NK3 antagonist SR142801 (100 nM) (both kind gifts from Dr Emonds-Alt, Sanofi Recherche, Montpellier, France).
7
Characterization of GV-58 Ion Channel Modulator
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GV-58 (1402821-41-3, CHEMBL2206242, SCHEMBL17628602, (2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol, C18H26N6OS, https://pubchem.ncbi.nlm.nih.gov/compound/71463101 , accessed on 1 December 2022), ω-conotoxin MVIID, and tetrodotoxin (TTX) were acquired from Alomone Labs (Genechain, Kaohsiung, Taiwan), while 4-aminopyridine, E-4031, nimodipine, ranolazine, retinoic acid, riluzole, and tetraethylammonium chloride (TEA) were from Sigma-Aldrich (Merck, Taipei, Taiwan). The stock solution of GV-58 was kept under −20 °C for long-term storage. We obtained culture media (e.g., Ham’s F-12 medium), fetal bovine or calf serum, horse serum, L-glutamine, and trypsin/EDTA from HycloneTM (Thermo Fisher Scientific, Tainan, Taiwan), and all other chemicals, including CsCl, CsOH, CdCl2, and EGTA, were of laboratory grade and supplied from standard sources.
The external solutions and pipette solutions used in this work are illustrated inTable 1 .
The external solutions and pipette solutions used in this work are illustrated in
8
Reagents for Neurophysiological Experiments
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AlexaFluor-594 hydrazide, AlexaFluor-594 dextran (10,000 MW), Fluo-4 and Fluo-4FF pentapotassium salts were purchased from Invitrogen. CsCl and NaHCO3 were obtained from ACP Chemicals, tetrodotoxin (TTX) from Alomone Labs, N-ethyllidocaine bromide (QX-314, Br-) and bicuculline from Tocris Bioscience, SP20 antibody from Santa Cruz Biotechnology (Cat# sc-65512, Lot# K1407, RRID:AB_1129364), paraformaldehyde from Electron Microscopy Sciences. We ordered NaCl, KCl, glucose, NaH2PO4, Na-pyruvate, myo-inositol, l -ascorbic acid, MgCl2, CaCl2, K-gluconate, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), HEPES, phosphocreatine, Na-ATP, Na-GTP, phosphocreatine di(tris) salt, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and Strychnine from Sigma-Aldrich.
9
Pharmacological Characterization of Synaptic Transmission
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All drugs were purchased from Sigma, except 6-methyl-2-(phenylethynyl) pyridine (MPEP) and K252a from Tocris, mBDNF, proBDNF (wild-type mouse, #B240) and tetrodotoxin (TTX) from Alomone, p75NTR antibody (AB-N01) from Advanced Targeting Systems. All drug stocks were freshly diluted to the desired concentrations. Final concentration of DMSO never exceeded 0.01 %. Extracellular solution in current-clamp and voltage-clamp experiments contained (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 1.8 MgSO4, 1.25 NaH2PO4, 10 glucose, 1.6 CaCl2 and pH was maintained at 7.4 by constant bubbling with carbogen (95 % O2, 5 % CO2). During persistent firing experiments synaptic transmission was blocked using kynurenic acid (2 mM) and picrotoxin (100 μM).
During mEPSCs recordings, tetrodotoxin (TTX, 1 μM) and picrotoxin (100 μM) were applied and spontaneous activity was recorded during 5 min with the resting membrane potential held at -70 mV. Intracellular solution for current-clamp and voltage-clamp was composed of (in mM): 120 K-gluconate, 10 HEPES, 0.2 EGTA, 20 KCl, 2 MgCl2, 7 diTrisP-Creatine, 4 Na2ATP and 0.3 NaGTP (pH adjusted to 7.3 with KOH).
During mEPSCs recordings, tetrodotoxin (TTX, 1 μM) and picrotoxin (100 μM) were applied and spontaneous activity was recorded during 5 min with the resting membrane potential held at -70 mV. Intracellular solution for current-clamp and voltage-clamp was composed of (in mM): 120 K-gluconate, 10 HEPES, 0.2 EGTA, 20 KCl, 2 MgCl2, 7 diTrisP-Creatine, 4 Na2ATP and 0.3 NaGTP (pH adjusted to 7.3 with KOH).
10
Multielectrode Array Neuronal Functional Assay
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48-well opaque-bottom MEA plates (Axion Biosystems), 16 electrodes per well, were coated with filter-sterilized 0.1% polyethyleneimine (PEI) solution in borate buffer pH 8.4 for 1 h at room temperature, washed 4 times with water, and dried overnight. The procedure followed as described [20 (link)] using Axion Biosystems with some modifications. After last reading of MEA plate at week six, wells were treated with three synaptic antagonists: Gamma-aminobutyric acid type A (GABA-A) receptor antagonist picrotoxin (PTX; Sigma) at 100 mM, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; Sigma) at 60 mM, and sodium ion channel antagonist tetrodotoxin (TTX; Alomone labs) at 1 mM (Fi g. 5 I). Plates were recorded for 10–15 min after addition of the antagonists. Culture media was replaced with CM2, and after 1 h recovery period, the final recording was taken. The weighted MFR represents the MFR per active electrode whereas burst is considered as a group of at least five spikes, each separated by an inter-spike interval (ISI) of no more than 100 ms. No non-active wells observed and therefore, no wells excluded in the analysis.
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