For in vivo experiments employing excitatory glutamatergic antagonists, these were purchased from Tocris (CNQX 0190, AP5 0106) and dissolved in standard 3.5 mM K+ ACSF on the day of experiment from concentrated aliquots to final concentrations of 0.8 mM and 1.6 mM for CNQX and AP5, respectively (Mateo et al., 2011 (link)). CNQX/AP5 was topically applied with 30 min incubation before evaluating the effect (Vinokurova et al., 2018 (link)). For cortical slice experiments, glutamatergic antagonists were dissolved in standard ACSF on the day of experiment from concentrated aliquots, to final concentrations of 20 μM and 50 μM for CNQX and AP5, respectively (Cotel et al., 2013 (link)). To block fast inhibitory GABAergic synaptic transmission in slice experiments, Gabazine was purchased from Tocris (SR 95531 hydrobromide) and dissolved in standard ACSF on the day of the experiment from concentrated aliquots to a final concentration of 10 μM (Cotel et al., 2013 (link)).
Sr 95531 hydrobromide
Manufactured by Bio-Techne
Sourced in United Kingdom
SR 95531 hydrobromide is a GABAA receptor antagonist. It selectively blocks the GABA-A receptor, which is the main inhibitory neurotransmitter receptor in the central nervous system.
Automatically generated - may contain errors
Lab products found in correlation
Gabazine, by Bio-Techne (4 mentions)
Rs cpp, by Bio-Techne (3 mentions)
Zd7288, by Bio-Techne (3 mentions)
Mni caged l glutamate, by Bio-Techne (2 mentions)
Bumetanide, by Merck Group (2 mentions)
Isoguvacine, by Merck Group (2 mentions)
Picrotoxin, by Bio-Techne (2 mentions)
Chlorprothixene hydrochloride, by Merck Group (2 mentions)
Nbqx disodium, by Bio-Techne (2 mentions)
Phenylephrine hydrochloride, by Bio-Techne (2 mentions)
Dexmedetomidine hydrochloride, by Bio-Techne (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Prazosin hydrochloride, by Bio-Techne (2 mentions)
Tetrodotoxin citrate, by Alomone (2 mentions)
Propranolol, by Merck Group (1 mentions)
Strychnine, by Merck Group (1 mentions)
L 741 626, by Bio-Techne (1 mentions)
Sch 39166 hydrobromide, by Bio-Techne (1 mentions)
Tetraethylammonium chloride, by Merck Group (1 mentions)
Quinpirole hydrochloride, by Bio-Techne (1 mentions)
13 protocols using sr 95531 hydrobromide
1
Glutamatergic and GABAergic Antagonist Protocols
2
Pharmacological Stimulation of Co-Cultures
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At DIV28, the co-cultures and the NS on MEAs were stimulated with 75 µM 4-Aminopyridine (4-AP; 0940; Tocris, Bristol, UK) or 30 µM SR-95531 hydrobromide (gabazine; 1262; Tocris, Bristol, UK). Before chemical stimulation, the cells on the MEAs were left to settle in the MEA2100-System preamplifier (Section 4.6 ) for five minutes to alleviate possible effects of mechanical disturbance from moving the MEAs from the incubator to the preamplifier. After that, spontaneous neuronal activity was recorded for five minutes. Subsequently, appropriate volumes of previously prepared stock solutions (4-AP: 10 mM; gabazine: 25 mM; both diluted in ultra-pure water) of the drugs were pipetted directly to the MEA wells to result in the final drug concentrations, and the cell cultures on the MEAs were immediately recorded for ten minutes. Finally, the whole medium reservoir was replaced with a fresh prewarmed cell medium. The same chemical stimulation replacement was applied on the 24-well plates and coverslips that were stimulated for approximately 24 h inside an incubator (Section 4.4 ).
3
Pharmacological Modulation of Neural Signaling
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All drugs were purchased from Sigma Aldrich (St. Louis, MO), or Tocris (Bristol, UK). SKF 81297 hydrobromide (Tocris, 1447) was applied either as puff (500 µM) or flow in (10 µM) depending on experiment type. All other drugs were bath applied: SCH-39166 hydrobromide (Tocris,2299), SCH-23390 hydrochloride (Tocris, 0925), SKF-83566 hydrobromide (Tocris, 1586), Strychnine (Sigma-Aldrich, 8753), Tetraethylammonium chloride (Sigma-Aldrich, 86614), TTX (Tocris Bioscience, 1069), (RS)-CPP (Tocris Bioscience, 0173), NBQX (Tocris Bioscience, 0373), SR 95531 hydrobromide (Tocris Bioscience, 1262), MNI-caged-L-glutamate (Tocris Biosciences 1490), L-741,626 (Tocris Bioscience, 1003), Pyr-3 (Tocris,3753), prazosin (Sigma-Aldrich, P7791), propranolol (Sigma-Aldrich, 40543), and quinpirole hydrochloride (Tocris Bioscience, 1061). For experiments requiring pharmacological agents dissolved in DMSO the concentration never exceeded 0.02% DMSO.
4
Pharmacological Modulation of Neuronal Signaling
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For in vitro experiments, SR 95531 hydrobromide (gabazine 5 μM, Tocris Bioscience, UK, Ref. 1262), NMDA (10 μM, Tocris Bioscience, UK, Ref. 0114), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (10 µM, NIH generous gift), GABA (10 μM, Sigma-Aldrich), isoguvacine (10 μM, Sigma-Aldrich, Ref. G002), DL -2-Amino-5-phosphonovaleric acid (40 µM, Sigma-Aldrich, Ref. A5282), and bumetanide (10 μM, Sigma-Aldrich, Ref. B3023) were directly added to the perfusion solutions. For ca experiments, slices were treated with bumetanide for 40 min before and during recordings. Cocktail of nicotinic receptor antagonists included mecamylamine hydrochloride (10 μM, Tocris Bioscience, Ref. 2843/10), methyllycaconitine citrate (0.1 μM, Tocris Bioscience, Ref. 1029/5), and dihydro-β-erythroidine hydrobromide (10 μM, Tocris Bioscience, Ref. 2349/10). For in vivo experiments, bumetanide pretreatment (3 mg kg−1) was given to mice in drinking water during 5 weeks.
5
Pharmacological Modulation of Neuronal Signaling
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All chemicals were purchased from VWR unless otherwise indicated. VU 10010 (M4-selective positive allosteric modulator), SR 95531 hydrobromide (Gabazine, GABAA antagonist), Baclofen (GABAB antagonist), QX314 chloride (intracellular sodium channel blocker), and AF-DX 116 (selective M2- muscarinic receptor antagonist) were obtained from Tocris Bioscience (Ellisville, Missouri) and 6, 7-Dinitroquinoxaline-2, 3-dione (DNQX, AMPA receptor antagonist), DL-2-Amino-5-phosphono pentanoic acid (APV, NMDA receptor antagonist) from Ascent Scientific (Bristol, U.K.). Biocytin (B-1592) was purchased from Life Technologies (Invitrogen).
6
Neurotransmitter Modulation in Neuronal Circuits
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The following drugs were used: glutamate (Sigma-Aldrich), γ-Aminobutyric acid (Sigma-Aldrich), MNI-caged-L -glutamate (Tocris Bioscience, Abingdon, UK), Tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel), SR 95531 hydrobromide (gabazine, Tocris Bioscience), Picrotoxin (Tocris Bioscience).
7
Uncaging Experiments with Synaptic Inhibitors
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D-AP5, NBQX disodium salt, tetrodotoxin, SR95531 hydrobromide, CGP55845, guangxitoxin, ZD7288 (all from Tocris) were dissolved in distilled water in stock solutions, aliquots were stored at −20 °C and used on the day of experiment. CdCl2 was purchased from Sigma-Aldrich. All inhibitors were applied in the bath. Note that the effective concentration of D-AP5 in the uncaging experiments is expected to be reduced at the stimulated dendritic region during puffing of drug-free MNI-glutamate solution.
8
Dissecting Neuronal Hyperactivity Mechanisms
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Chemicals inhibiting specific ion channels were introduced in the bath/recording buffer to compare different physiological components possibly contributing to the neuronal hyperactivity. To block all fast-synaptic excitatory transmission, 3-[(±)2-carboxypiperazin-4yl] propyl-1-phosphate (CPP, NMDA receptor antagonist, 20 μM; Tocris Bioscience, United Kingdom) and 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX, AMPA/kainate (non-NMDA) receptor antagonist, 20 μM, diluted in DMSO; Alomone Labs, Israel] were introduced in the bath. SR95531 hydrobromide or Gabazine (GABAA receptor antagonist, 10 μM, Tocris Bioscience, United Kingdom) was used to block inhibitory synaptic transmission. Cd2+ (CdCl2, 100 μM; Sigma-Aldrich, United States) was used to depress synaptic transmission and block inward calcium-selective current, isolating the outward K+ current. To block voltage-dependent K+ channels, intracellular administration of cesium was done by replacing potassium gluconate with cesium gluconate in the internal solution while maintaining the same osmolarity.
9
In vivo and in vitro imaging of neuronal activity
10
Pharmacological Dissection of Synaptic Transmission
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Bicuculline-methiodide (10 μM; Abcam, Cambridge, MA, USA) or SR95531-hydrobromide (10 μM; Tocris, Ellisville, MO, USA) was present in the saline for all experiments to block GABAA receptor mediated synaptic transmission. After recording control responses, 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (20 μM; ZD7288; Tocris Bioscience, Ellisville, MO, USA) was washed in for 10 min to block HCN channels. ZD7288 was applied at a 10 μM concentration in a set of control experiments in order to rule out dose-dependent, off-target effects. In another set of control experiments, 20 μM ZD7288 was added to the normal K-gluconate internal solution for cell-specific, post-synaptic HCN channel inhibition. Tetrodotoxin-citrate (1 μM; Sigma, St. Louis, MO, USA) was used to block AP mediated synaptic transmission for the analysis of mEPSCs. All drugs were bath applied unless otherwise stated, with each neuron serving as its own control.
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