Diazepam

Manufactured by Bio-Techne

Sourced in United Kingdom

Diazepam is a laboratory equipment product manufactured by Bio-Techne. It is a chemical compound used in various research and analytical applications.

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16 protocols using diazepam

Detailed Protocol of Cell Culture Reagents

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1-Aminobenzotriazole, ammonium chloride, ciprofoxacin, clarithromycin, dimethyl sulphoxide, Imipramine, lactate dehydrogenase activity assay kit, monensin sodium salt, nigericin sodium salt, trypan blue 0.4% and verapamil hydrochloride were all from Sigma Aldrich Ltd., Dorset, UK. Budesonide, ipratropium bromide, fenoterol, formoterol, terbutaline and tiotropium bromide were all supplied by GlaxoSmithKline, UK. Chloroform and formaldehyde 37–41% were from Fisher Scientific, Loughborough, UK. Further chemicals include diazepam (Tocris Bioscience, Bristol, UK), methanol (VWR, UK), midazolam (Hoffman La Roche, Switzerland), Pierce BCA protein assay kit (Thermo Scientific, Loughborough, UK) and Lysotracker® Red DND-99 (Life Technologies, Paisley, UK). Reagents include bovine serum albumin and penicillin-streptomycin (Sigma Aldrich Ltd., Dorset, UK), collagen Type I rat tail (BD Biosciences, Oxford, UK), Dulbecco’s phosphate buffered saline and heat-inactivated foetal bovine serum (Life Techonolgies, Paisley, UK) and Kaighn’s modification of Ham’s F12 (Ham’s F12K) medium (American Tissue Culture Collection (ATCC), Mannasas, VA, USA).

Ufuk A., Somers G., Houston J.B, & Galetin A. (2015). In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383. Pharmaceutical Research, 32(12), 3937-3951.

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Patch-clamp analysis of GABA receptor

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Whole cell voltage-clamp recordings were made from cells transiently transfected with the constructs used in structural analysis. For the patch-clamp experiments, adherent HEK293S GnTI⁻ cells were transiently transfected with pEZT-based plasmids 2-3 days before recording. Each 35 mm dish of cells was transfected with the DNA of α1 subunit, β2 subunit and γ2 subunit in a ratio of 1:1:4 to ensure the incorporation of γ2 subunit. At the time of transfection, cells were moved to 30 °C. On the day of recording, cells were washed with bath solution, which contains (in mM): 140 NaCl, 2.4 KCl, 4 MgCl₂, 4 CaCl₂, 10 HEPES pH 7.3 and 10 glucose. Borosilicate pipettes were pulled and polished to a resistance of 2-4 MΩ. The pipette solution contained (in mM): 150 CsCl, 10 NaCl, 10 EGTA, 20 HEPES pH 7.3. Cells were clamped at −75 mV. The recordings were made with an Axopatch 200B amplifier, low pass filtered at 2 kHz and digitized at 5 kHz using the Digidata 1440A and pClamp 10 software (Molecular Devices). The GABA, flumazenil, diazepam (Tocris Bioscience), and bicuculline (Sigma-Aldrich) solutions were prepared in bath solution. Stock solution of 1 M GABA was prepared in water; stock solutions of 100 mM bicuculline, 10 mM diazepam and 10 mM flumazenil were prepared in DMSO. Solution exchange was achieved using a gravity driven RSC-200 rapid solution changer (Bio-Logic).

Zhu S., Noviello C.M., Teng J., Walsh RM J.r., Kim J.J, & Hibbs R.E. (2018). Structure of a human synaptic GABA-A receptor. Nature, 559(7712), 67-72.

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Pharmacological Profiling of GABA Receptor Subunits

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Electrophysiological recordings of HEK293 cells stably expressing GABA_ARs were performed in a whole-cell, voltage-clamp mode. The chamber containing coverslips with the cell line was continuously superfused at a flow rate of 1.8 ml/min with the extracellular medium composed of 130 mM NaCl, 4 mM KCl, 10 mM HEPES, 20 mM NaHCO₃, 10 mM glucose, 1 mM MgCl₂, and 2 mM CaCl₂, and was equilibrated with 5% CO₂/95% O₂ and maintained at room temperature (~21–25°C). The electrodes were filled with an intracellular solution containing (in mM), 130 KCl, 3 NaCl, 4.5 phosphocreatine, 10 HEPES, 1 EGTA, 3.5 Na-ATP, 0.45 Na-GTP, and 2 MgCl₂ (adjusted to pH 7.2 with KOH, 290–300 mOsmol/l), and had a final resistance of 3–8 MΩ. To test the target selectivity of α5-SOP002, the responsiveness to applied GABA was investigated and measured in HEK293 cells stably expressing either, α5β2γ2, α1β2γ2 or α2β2γ2 subunits of GABA_ARs. The pharmacological properties of the expressed receptors were investigated by puffer-application of GABA (1 μM; Tocris Bioscience, UK) and subsequent bath-application of α5-SOP002 (0.5–1 μM), followed by diazepam (1 μM, Tocris Bioscience, UK). The change in voltage after the GABA puff application response was recorded. The statistical test used was one-way ANOVA with a 95% confidence interval.

Petrache A.L., Khan A.A., Nicholson M.W., Monaco A., Kuta-Siejkowska M., Haider S., Hilton S., Jovanovic J.N, & Ali A.B. (2020). Selective Modulation of α5 GABAA Receptors Exacerbates Aberrant Inhibition at Key Hippocampal Neuronal Circuits in APP Mouse Model of Alzheimer’s Disease. Frontiers in Cellular Neuroscience, 14, 568194.

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Investigating Diazepam and Flumazenil Effects

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The methods are identical to those published by the same author previously (
Humble, 2016a (link)), with the exception of the drugs Diazepam and flumazenil, which were not used in the previous study. Diazepam and flumazenil were purchased (Tocris, Bristol UK) and prepared as concentrated stock solutions in dimethyl sulfoxide before being added to the artificial extracellular solution as per the previous study (
Humble, 2016a (link)).

, & Humble S.R. (2018). Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis.. F1000Research, 6, 506.

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Electrophysiological Experiments with RDX

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RDX (Hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine) was purchased as certified reference material (Cerilliant® from MilliporeSigma) in a 1‐mg/mL solution in acetonitrile (1 mL ampules, chromatographic purity 99.9%). For electrophysiological experiments, the acetonitrile was evaporated and RDX immediately reconstituted in 1 mL DMSO rendering 0.1 molar stocks. RDX waste was treated with nitric acid and disposed of using the waste accumulation program at UC Davis. Bicuculline, picrotoxinin, and GABA were purchased from MilliporeSigma. Diazepam was purchased from Tocris Bioscience.

Pressly B., Lee R.D., Singh V., Pessah I.N, & Wulff H. (2022). The seizure‐inducing plastic explosive RDX inhibits the α1β2γ2 GABAA receptor. Annals of Clinical and Translational Neurology, 9(5), 600-609.

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Preparatory Protocol for Electrophysiology

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Picrotoxinin (PTX), fipronil, bicuculline, propofol, salicylidene salicylhydrazide, zinc chloride, GABA, dexamethasone, zeocin, and geneticin were purchased from Sigma Aldrich (St. Louis, MO, United States). Diazepam, allopregnanolone, and DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) were purchased from Tocris Bioscience (Bristol, United Kingdom). TETS was synthesized in the laboratory of Dr. Bruce Hammock, University of California, Davis, CA (Zhao et al. 2014 (link)). 10 mM stocks of GABA were made fresh daily using Ringer solution (see below for composition). 10 mM stocks of PTX and TETS were prepared in DMSO and diluted down into Ringer solution immediately before application onto the cell. Both TETS and PTX waste were treated with nitric acid and disposed of using the waste accumulation program at UC Davis.

Pressly B., Nguyen H.M, & Wulff H. (2017). GABAA receptor subtype selectivity of the proconvulsant rodenticide TETS. Archives of Toxicology, 92(2), 833-844.

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Pharmacological Agents for Behavioral Studies

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Lactic acid and ketorolac tris salt were purchased from Sigma Chemical Co (St. Louis, MO) and prepared in sterile water and sterile saline, respectively. Diazepam was purchased from Tocris Bioscience (Minneapolis, MN), and KRM-II-81 (5-(8-ethynyl-6-(pyridine-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl) oxazole) was synthesized at the University of Wisconsin-Milwaukee and generously provided by Dr. James Cook. Diazepam and KRM-II-81 were dissolved in 2:1:7 DMSO:emulphor:saline. All drugs were injected i.p. in a volume of 1 ml/kg.

Moerke M.J., Li G., Golani L.K., Cook J, & Negus S.S. (2019). Effects of the α2/α3-subtype-selective GABAA receptor positive allosteric modulator KRM-II-81 on pain-depressed behavior in rats: Comparison with ketorolac and diazepam. Behavioural pharmacology, 30(5), 452-461.

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Internalization of GABAA Receptor Subunits

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Cell-surface receptors were labelled in living cultured neurons or in α1β2-HEK293 cells transiently transfected with the myc-γ2 cDNA, as described previously 19 (link). Briefly, coverslips were incubated with ice-cold Buffer A (mM: 150 NaCl; 3 KCl; 2 MgCl₂; 10 HEPES, pH 7.4; 5 Glucose), containing 0.35 M sucrose for 5 minutes, followed by incubation with mouse anti-β_2/3 antibody (MAB341-MerckMillipore) for 30 minutes at 4°C in Buffer A containing 0.35 M sucrose, 1 mM EGTA and 1% BSA. Cells were further incubated at 37°C in Buffer A containing 1 mM CaCl₂ and 5 µg/ml leupeptin, in the absence or presence of diazepam (1 µM, Tocris), for 1 hour to allow internalization of the labelled β_2/3 subunit-containing GABA_ARs. Cells were fixed with 4% paraformaldehyde/4% sucrose/PBS (PFA/PBS) and processed for immunolabelling with anti-mouse Alexa-555 antibodies overnight at 4°C. Cells were subsequently permeabilized and labelled with anti-mouse Alexa-488 antibodies (Supplementary Table S1) for 1 hour at room temperature. Samples were imaged using a Zeiss LSM 710 confocal microscope as above.

Nicholson M.W., Sweeney A., Pekle E., Alam S., Ali A.B., Duchen M, & Jovanovic J.N. (2018). Diazepam-induced loss of inhibitory synapses mediated by PLCδ/ Ca2+/calcineurin signalling downstream of GABAA receptors. Molecular Psychiatry, 23(9), 1851-1867.

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Glutamatergic and GABAergic Receptor Modulation

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Drugs acting at glutamatergic and GABAergic receptors were used at the following concentrations: 50 µmol/L D‐2‐amino‐5‐phosphonovalerate (AP5) (Hello Bio, Bristol, UK); 20 µmol/L NBQX disodium salt (Hello Bio, Bristol, UK); 10 μmol/L Gabazine (Hello Bio, Bristol, UK); 3 µmol/L Diazepam (Tocris, Abingdon, UK); 5 μmol/L CGP‐55845 (Hello Bio, Bristol, UK).

Codadu N.K., Graham R.T., Burman R.J., Jackson‐Taylor R.T., Raimondo J.V., Trevelyan A.J, & Parrish R.R. (2019). Divergent paths to seizure‐like events. Physiological Reports, 7(19), e14226.

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Preparation of Experimental Solutions

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Capsaicin (Sigma Chemical, St. Louis, MO, USA) was dissolved in the vehicle containing 10% ethanol and 10% Tween 80, sonicated for 5 min, and then further diluted (1:100) in an artificial cerebrospinal fluid solution (aCSF) as a stock solution stored at 4°C. The aCSF consisted of the followings (in mm): 117 NaCl, 4.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11.4 dextrose bubbled with 95% O₂/5% CO₂, pH 7.4. Compound 6 was synthesized as reported previously (Varagic et al., 2013a (link)). Loreclezole, Ro15–4513, and diazepam (under the approval from Taiwan Food and Drug Administration, the Ministry of Health and Welfare, Taiwan) were purchased from Tocris Bioscience (Bristol, UK) and furosemide from Sigma-Aldrich (St. Louis, USA). All compounds were dissolved in a vehicle containing 20% DMSO, 20% Cremophor^® EL (polyoxyethylene castor, Sigma-Aldrich) and 60% normal saline.

Fan P.C., Lai T.H., Hor C.C., Lee M.T., Huang P., Sieghart W., Ernst M., Knutson D.E., Cook J, & Chiou L.C. (2018). The α6 Subunit-Containing GABAA Receptor: A Novel Drug Target for Inhibition of Trigeminal Activation. Neuropharmacology, 140, 1-13.

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