After incubating for 30 min in either 0.01% DMSO or 15 µM PW201, slices were transferred to a recording chamber perfused with continuously oxygenated and heated standard aCSF. Somatic recordings of MSNs were then performed using electrodes filled with an internal solution comprised of the following salts: 145 mM K-gluconate; 2 mM MgCl2; 0.1 mM EGTA; 2.5 mM Na2ATP; 0.25 mM Na2GTP; 5 mM phosphocreatine; and 10 mM HEPES (pH = 7.2 and osmolarity = 290 mOsm; all salts were purchased from Sigma-Aldrich). After GΩ formation and entry into the whole-cell configuration, the amplifier was switched to I = 0 mode for approximately 1 min to determine the resting membrane potential before switching to current-clamp mode to assess intrinsic excitability. During this 1 min interval in I = 0 mode, the following cocktail of synaptic blockers was perfused to halt changes in excitability driven by synaptic activity: 20 µM bicuculine; 20 µM NBQX; and 100 µM AP5 (synaptic blockers purchased from Tocris, Bristol, UK). To assess intrinsic excitability, evoked APs were measured in response to a range of current injections from −20 to +150 pA. Current steps were 800 ms in duration, and the change in the injected current between steps was 10 pA.
Bicuculine
Manufactured by Bio-Techne
Sourced in United Kingdom
Bicuculine is a laboratory reagent used in scientific research. It functions as a competitive antagonist of the GABA-A receptor, a key inhibitory neurotransmitter receptor in the central nervous system.
Automatically generated - may contain errors
Lab products found in correlation
K gluconate, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Risperidone, by Merck Group (1 mentions)
Spermine, by Merck Group (1 mentions)
Bapta, by Merck Group (1 mentions)
Fk506, by Merck Group (1 mentions)
Kn 62, by Merck Group (1 mentions)
Ro 31 8220, by Bio-Techne (1 mentions)
Iem 1460, by Bio-Techne (1 mentions)
U0126, by Merck Group (1 mentions)
Nicotine, by Merck Group (1 mentions)
Strychnine, by Bio-Techne (1 mentions)
Glycine, by Merck Group (1 mentions)
Bicuculine, by Merck Group (1 mentions)
Axopatch 1d patch clamp amplifier, by Molecular Devices (1 mentions)
6 protocols using bicuculine
1
Measuring Intrinsic Excitability in MSNs
2
Risperidone Treatment in Behavioral Tests
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Risperidone (Sigma-Aldrich, St. Luis, MO, USA, R3030, 0.01 mg/kg) dissolved with saline was administered by intraperitoneal (i.p.) injections 30 min before each behavioral test. Especially, administration was performed on each day of the novel object recognition and fear-conditioning test. Saline was injected to the control mice as a vehicle. Bicuculine and NBQX were purchased from Tocris Bioscience (Bristol, UK).
3
Pharmacological Dissection of Synaptic Plasticity Mechanisms
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The following reagents (final concentration) were included in this study. DHβE (1 μM), MLA (10 nM), APV(50 μM), IEM-1460(50 μM), Ro318220(10 μM), Bicuculine (10 μM), TTX(1 μM) were from TOCRIS. (−)-nicotine (N3876, covered with dark paper when in use), U0126 (10 μM), FK506 (10 μM), KN62 (15 μM), H89 (10 μM), BAPTA (10 mM) and spermine (100 μM) were from Sigma. Lipid solvent were made in stock solutions in DMSO (1:1000–2000), while the same dilution of DMSO were used in control solutions. Peptides that were designed to block the phosphorylation of GluA1 at the PKA (S845), CAMKII (S831) and PKC (S816/818) sites were synthesized and purified by Pepmic Co., Ltd (Suzhou, China). The sequences of PKA S845 was TLPRNSGAG (-SGAG), scrambled control was NRPGGTLSA (-TLSA). That of CaMKII S831 was QSINEAIRTSTLPRN (-LPRN), scrambled control was NLIITEQRPSSNART (-NART). And that of PKCS816/818 was EFCYKSRSESKRMKGFC (-KGFC), scrambled control was RGMKESSKKCCRSFFYE (-FFYE). They were all diluted in internal solution with a final concentration of 200 μM.
4
Isolated NMDAR mEPSC Measurement
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To obtain NMDAR mEPSCs, dual AMPAR-NMDAR mEPSCs were measured in the presence of 0.5 µM TTX, 10 µM bicuculine, 10 µM strychnine (all from Tocris) and 10 µM glycine (Sigma). Subsequently, 50 µM AP-5 was added and glycine was washed to measure AMPAR mEPSCs from the same cell. To get average NMDAR mEPSC, AMPAR mEPSCs from one cell were aligned, averaged and subtracted from the aligned (AMPA peak was used to align) and averaged dual AMPAR-NMDAR mEPSCs from the same cell28 (link). The amplitude of average NMDAR mEPSC was found as the mean of the data in the interval 7 to11 ms after the onset. Different intracellular solution was used in this experiment to minimize baseline noise (in mM): 125 gluconic acid, 15 CsCl, 5 EGTA, 10 Hepes, 3 MgCl2, 0.5 CaCl2 and 2 ATP-Mg salt (pH-adjusted to 7.2 with CsOH).
5
Electrophysiological Assessment of Synaptic Transmission in APP/PS1 Mice
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Spontaneous and miniature EPSCs were studied in hippocampal CA1 pyramidal neurons in acute slices with no CA3-CA1 cut, prepared from APP/PS1 and WT littermate mice brains as described above. The EPSCs were recorded with the intracellular solution containing (in mM): 140 potassium gluconate, 10 HEPES, 20 KCl, 4 Mg-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine; pH was adjusted to 7.2–7.4 using 1 M KOH (280–290 mOsmol/L; [25 (link)]). Spontaneous EPSCs were studied in the presence of bicuculine (10 µM, Sigma Aldrich) and miniature EPSCs were examined in the presence of bicuculine (10 µM) and tetrodotoxin (TTX, 1 µM, Tocris) in the extracellular aCSF [67 (link)]. EPSCs were recorded at -70 mV holding potential, for the period of 10 min; 250 events from each sweep were used for data analyses. The temperature of external solution in the recording chamber was maintained at 34–36 °C (aCSF perfusion rate- 2.4 mL/min) for EPSC recordings. Data were analyzed manually using the ‘Mini Analysis program’ (version 6.0.7, Synaptosoft, Decatur, GA, USA).
6
Patch-Clamp Analysis of Neuronal Synaptic Currents
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All experiments were performed in the whole-cell configuration of patch-clamp mode using neurons cultured for 19 DIV. Typical resistances of pipettes used for recordings were $5 MU when filled with internal solution composed of the following: 110 mM CsCl, 4 mM MgCl 2 , 1 mM EGTA, 10 mM HEPES, 3 mM Na 2 ATP, and 1 mM NaGTP (pH 7.2). The external solution contained 130 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 15 mM glucose, and 15 mM HEPES (pH 7.4). Recordings of mEPSCs were carried out at a holding potential of À70 mV in the presence of 50 mM bicuculine (Tocris) and 1 mM TTX (Tocris). Recordings of spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were obtained at À70 mV in the presence of 5 mM CNQX (Tocris) and 1 mM TTX.
All salts were from Sigma-Aldrich. Before the addition of glucose and CaCl 2 , the osmolality of the external solution was adjusted to 290 mOsm/kg. All experiments were performed at room temperature (23 C). Recordings were made using an Axopatch-1D patch-clamp amplifier (Molecular Devices) under the control of an ITC-18 board (Instrutech) driven by WCP software (Dr. John Dempster, University of Strathclyde). Recordings were acquired at 10 kHz and low-pass-filtered to 5 kHz. Analysis of mEPSCs and mIPSCs was carried out using macros written in Igor Pro software.
All salts were from Sigma-Aldrich. Before the addition of glucose and CaCl 2 , the osmolality of the external solution was adjusted to 290 mOsm/kg. All experiments were performed at room temperature (23 C). Recordings were made using an Axopatch-1D patch-clamp amplifier (Molecular Devices) under the control of an ITC-18 board (Instrutech) driven by WCP software (Dr. John Dempster, University of Strathclyde). Recordings were acquired at 10 kHz and low-pass-filtered to 5 kHz. Analysis of mEPSCs and mIPSCs was carried out using macros written in Igor Pro software.
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