IC50: 3 μM (GABAA)[3]

Maximum response to GABA is achieved at 1 μM and 3 μM of bucculline methyl chloride. In Xenopus laevis oocytes expressing human α1β2γ2L GABAA receptors, bucculline methyl chloride appears to parallel shift the GABA dose-response curve to the right without lowering the maximal GABA response [3]. This suggests that it is a competitive antagonist. In Xenopus laevis oocytes, bicuculline methyl chloride (1-100 μM; applied as an external patch; 2 minutes) efficiently inhibits Apamin-insensitive K2 channels and Apamin-sensitive small calcium-activated potassium channel (SK2) currents. sensitive current in SK1 [4].

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The sesquiterpene trilactone bilobalide is one of the active constituents of the 50:1 Ginkgo biloba leaf extract widely used to enhance memory and learning. Bilobalide was found to antagonise the direct action of gamma-aminobutyric acid (GABA) on recombinant alpha(1)beta(2)gamma(2L) GABA(A) receptors. The effect of bilobalide on the direct action of GABA at alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp method was evaluated and compared with the effects of the classical GABA(A) receptor competitive antagonist BicucuLline and noncompetitive antagonist picrotoxinin. Bilobalide (IC(50)=4.6+/-0.5 microM) was almost as potent as bicuculline and pictrotoxinin (IC(50)=2.0+/-0.1 and 2.4+/-0.5 microM, respectively) at alpha(1)beta(2)gamma(2L) GABA(A) receptors against 40 microM GABA (GABA EC(50)). While bilobalide and picrotoxinin were clearly noncompetitive antagonists, the potency of bilobalide decreased at high GABA concentrations suggesting a component of competitive antagonism. [3]

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Small-conductance calcium-activated potassium channels (SK channels) are gated solely by intracellular calcium ions and their activity is responsible for the slow afterhyperpolarization (AHP) that follows an action potential in many excitable cells. Brain slice studies commonly employ a methyl derivative of BicucuLline (bicuculline-m), a GABAA (gamma-aminobutyric acid) receptor antagonist, to diminish the tonic inhibitory influences of GABAergic synapses, or to investigate the role of these synapses in specialized neural networks. However, recent evidence suggests that bicuculline-m may not be specific for GABAA receptors and may also block the slow AHP. Therefore, the effects of bicuculline-m on cloned apamin-sensitive SK2 and apamin-insensitive SK1 channels were examined following expression in Xenopus oocytes. The results show that at concentrations employed for slice recordings, bicuculline-m potently blocks both apamin-sensitive SK2 currents and apamin-insensitive SK1 currents when applied to outside-out patches. Apamin-insensitive SK1 currents run down in excised patches. The potency of bicuculline-m block also decreases with time after patch excision. Site-directed mutagenesis that changes two residues in the outer vestibule of the SK1 pore that confers apamin sensitivity also reduces run down of the current in patches, and endows stable sensitivity to bicuculline-m indistinguishable from SK2. Therefore, the use of bicuculline-m in slice recordings may mask apamin-sensitive slow AHPs that are important determinants of neuronal excitability. In addition, bicuculline-m-insensitive slow AHPs may indicate that the underlying channels have run down[4].

In mice, clonic convulsions are induced in a dose-dependent manner by bucculline methyl chloride (1.25-3 mg/kg; subcutaneous injection); these convulsions are exacerbated by administration of morphine, a μ-opioid receptor agonist [1]. Mice with a CD50 (convulsive dose) of 2.2 mg/kg for clonus and 2.4 mg/kg for tonus are susceptible to generalized seizures when injected subcutaneously with bicuculline methyl chloride (1.5-3.2 mg/kg). In order to prevent seizures caused by bicuculline methyl chloride, an intravenous injection of the NMDA antagonists MK-801, CPP, and CGS 19755 can be used as a pretreatment [2].

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The present study investigated the effects of micro-, delta- and kappa-opioid receptor agonists on seizures produced by blockade of gamma-aminobutyric acid (GABA)-mediated synaptic transmission in the mouse. The selective GABA(A) receptor antagonist BicucuLline (1.25-3 mg/kg) given subcutaneously caused dose-dependent clonic-tonic convulsions. These convulsions were potentiated by the prototypic mu-opioid receptor agonist morphine given subcutaneously 20 min prior to a subconvulsive dose of bicuculline. The potentiation by morphine was completely reversed by pretreatment intraventricularly with the selective mu-opioid receptor antagonist beta-funaltrexamine (0.5 microgram/mouse). Pretreatment intraventricularly with the selective delta-opioid receptor agonists 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12, 12abeta-octahydro-quinolino[2,3,3-g]isoquinoline ((-)TAN-67) or [D-Pen(2,5)]-enkephalin (DPDPE) showed a dose-dependent increase in the incidence of convulsions. Pretreatment with naltrindole (2 mg/kg, s.c.), a selective delta-opioid receptor antagonist, abolished the enhancement of the BicucuLline-induced convulsions by DPDPE. In contrast, pretreatment with the selective kappa-opioid receptor agonist U-50,488H (0.6-80 mg/kg, subcutaneously or 25-100 microgram/mouse, intraventricularly) produced a dose-dependent suppression of the bicuculline-induced convulsions. The inhibitory effect of U-50,488H was completely blocked by pretreatment subcutaneously with nor-binaltorphimine (5 mg/kg), a selective kappa-opioid receptor antagonist. This study demonstrates that activation of both mu- and delta-opioid receptors increases the incidence of convulsions produced by blockade of GABA-mediated synaptic transmission, while stimulation of kappa-opioid receptors has an anticonvulsive effect. [1]

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The effects of excitatory amino acid antagonists on convulsions induced by intracerebroventricular (i.c.v.) or systemic (s.c.) administration of the gamma-aminobutyric acidA (GABAA) antagonist bicuculline (BIC) were tested in mice. 3-[+/-)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP), 2-amino-7-phosphonoheptanoate (AP7) and (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate (MK-801) were used as representatives of N-methyl-D-aspartate (NMDA) antagonists. gamma-D-Glutamylaminomethylsulphonate (gamma-D-GAMS) typified a preferential kainate (KA) antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) represented a preferential quisqualate (QA) antagonist, and kynurenic acid (KYNA) was used as a mixed NMDA/KA antagonist. BicucuLline methiodide (BMI) induced clonic convulsions following i.c.v. administration with a CD50 of 0.183 nmol (range 0.164-0.204). The excitatory amino acid antagonists blocked clonic seizures induced by BMI in the dose of 0.224 nmol (approximately CD97) when coinjected into the lateral ventricle. CPP (ED50 0.0075 nmol) was the most potent anticonvulsant and was followed by AP7 (0.182 nmol), MK-801 (0.22 nmol), gamma-D-GAMS (0.4 nmol), KYNA (1.7 nmol) and CNQX (5.17 nmol). Muscimol (MSC), the GABAA agonist, blocked BMI-induced seizures with an ED50 of 0.25 nmol. Systemic (s.c.) administration of BIC induced in mice generalized seizures with a CD50 of 2.2 mg/kg (range 1.9-2.5) for clonus and CD50 of 2.4 mg/kg (range 2.2-2.7) for tonus.2+ the pathogenesis of seizures triggered by bicuculline in mice [2].

Electrophysiogical recording [3] Receptor activity was measured with two-electrode voltage-clamp techniques 2–8 days after injection. Recording microelectrodes were fabricated with a micropipette puller and filled with 3 M KCl solution. Oocytes were placed in a cell bath and voltage clamped at −60 mV. Cells were continuously superfused with ND96 buffer. The currents elicited in response to the application of drugs were recorded using a Geneclamp 500 amplifier, a Mac Lab 2e recorder, and Chart version 3.5.2 program on a Macintosh Quadra 605 computer. Drugs were tested for direct activation of GABA at GABAA receptors. For measurements of inhibitory action of drugs on receptor activation, drugs were added to the buffer solution containing GABA at the concentration producing 10%, 50%, 75%, 90% and 100% of the effect (GABA EC10, EC50, EC75, EC90 and EC100) at the receptors for constructing GABA inhibition dose–response curves. The same procedure, but with a fixed concentration of antagonists and increasing concentrations of GABA, was applied to construct GABA dose–response curves. A washout period of 3–5 min was allowed between each drug application to prevent receptor desensitisation.

Expression of α1β2γ2L GABAA receptors in Xenopus laevis oocytes [3]
Female X. laevis were anaesthetised with 0.17% ethyl 3-aminobenzoate in saline and a lobe of the ovaries surgically removed. The lobe of ovaries was rinsed with OR-2 buffer that contained 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2·6H2O, 5 mM HEPES, pH 7.4, and suspended in a solution of collagenase A (2 mg/ml in OR-2) for 2 h to separate oocytes from connective tissues and follicular cells. Released oocytes were then thoroughly rinsed in ND96 buffer supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline and 50 μg/ml gentamycin, and stage V to VI oocytes were collected. Human α1, β2 and γ2L cDNAs subcloned in pcDM8 were linearised using the restriction enzyme NOT1. Linearised plasmids containing α1, β2 and γ2L cDNAs were transcribed using T7 RNA Polymerase and capped with 5,7-methylguanosine using the “mMESSAGE mMACHINE” kit. Ten nanograms per 50 nl of a 1:1:1 mixture of α1, β2 and γ2L cRNAs were injected using a 15–20 μm diameter tip micropipette into the cytoplasm of individual defolliculated oocytes by using a Nanoject injector. The oocytes were incubated in ND96 buffer at 16 °C in an orbital shaker with a twice-daily change of buffer.

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Behavioral observations [1]
Mice were injected s.c. with several doses of BicucuLline. After s.c. injection, the tonic–clonic convulsions were observed for 10 min. The proportion of mice exhibiting convulsions at each dosage of bicuculline within 10 min after the injection was calculated. Groups of mice were injected s.c. or i.c.v. according to the method of Haley and McCormick [5] with opioids prior to the injection of bicuculline. BicucuLline was dissolved in saline acidified to pH 3 using 0.1 N HCl.

[1]. Effects of differential modulation of mu-, delta- and kappa-opioid systems on bicuculline-induced convulsions in the mouse. Brain Res. 2000 Apr 17;862(1-2):120-6.

[2]. Excitatory amino acid antagonists protect mice against seizures induced by bicuculline. Brain Res. 1990 Apr 23;514(1):131-4.

[3]. Bilobalide, a sesquiterpene trilactone from Ginkgo biloba, is an antagonist at recombinant alpha1beta2gamma2L GABA(A) receptors. Eur J Pharmacol. 2003;464(1):1-8.

[4]. Bicuculline block of small-conductance calcium-activated potassium channels. Pflugers Arch. 1999 Aug;438(3):314-21.

The present study has clearly demonstrated that morphine can enhance the BicucuLline-induced convulsions in a dose-dependent manner. This effect was completely reversed by the addition of the selective μ-opioid receptor antagonist β-FNA, indicating that the proconvulsive effect of morphine could be fully explained by a specific action at μ-opioid receptors. It has been widely recognized that morphine inhibits the release of GABA, resulting in the suppression of GABAAergic synaptic transmission in some brain regions. On the basis of the current findings together with the previous reports, it is likely that activation of μ-opioid receptors by morphine leads to the suppression of GABAAergic synaptic transmission in the presence of bicuculline, resulting in the enhancement of the bicuculline-induced convulsions.
Like morphine, the selective δ-opioid receptor agonists (−)TAN-67 and DPDPE, when given i.c.v., potentiated the BicucuLline-induced convulsions. This effect was completely blocked by the selective δ-opioid receptor antagonist NTI, confirming the δ-opioid receptor specificity. Although more anatomical and biochemical evidence is needed, these findings suggest that, like the μ-opioid receptor-dependent response, stimulation of the central δ-opioid receptors presynaptically modulates the release of GABA, resulting in the suppression of the GABAAergic synaptic transmission.
The mice treated with the selective κ-opioid receptor agonist U-50,488H displayed dose-dependent reductions in the expression of the BicucuLline-induced convulsions. The effect of blockade of κ-opioid receptors by nor-BNI on the anticonvulsant action of U-50,488H suggests that it is selectively mediated by κ-opioid receptors. U-50,488H has been shown to be an anticonvulsant against electrically induced seizures. It is well documented that κ-opioid receptor agonists affect mostly Ca2+ channels, resulting in blockade of Ca2+ entry. Although the mechanism of the anticonvulsive effect with U-50,488H is presently unclear, one possibility that should be mentioned is that postsynaptically localized κ-opioid receptors may contribute to the inhibition of excitability induced by postsynaptic blockade of GABAAreceptors through the reduction of Ca2+ entry.
In conclusion, the present study in mice showed that μ- and δ-opioid receptor agonists potentiated the BicucuLline-induced clonic–tonic convulsions, whereas the κ-opioid receptor agonist suppressed the convulsions induced by bicuculline. The effects of each agonist were completely reversed by the addition of the representative selective antagonist, confirming the receptor specificity of each agonist-induced effect. These findings may have implications for our understanding of the effects of differential modulation of μ-, δ- and κ-opioid peptide systems on the GABAAergic synaptic transmission.[1]

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BicucuLline is a competitive antagonist of GABAA receptors (Akaike et al., 1985). The competitive antagonism of bicuculline and noncompetitive antagonism of picrotoxinin at GABAA receptors are also exemplified at the human α1β2γ2L subunit combination. At α1β2γ2L GABAA receptors, bicuculline displayed the general property of the competitive antagonist, producing a parallel shift of GABA concentration–effect curves and having no effect on the maximal response of GABA. [3]

C21H20CLNO6

417.84

417.098

38641-83-7

Bicuculline methiodide;40709-69-1;Bicuculline;485-49-4;Bicuculline methobromide;66016-70-4

10047593

Light yellow to green yellow solid powder

0

7

1

29

655

2

[Cl-].O1C(C2C3=C(C=CC=2[C@@H]1[C@@H]1C2C=C4C(=CC=2CC[N+]1(C)C)OCO4)OCO3)=O

RLJKFAMYSYWMND-GRTNUQQKSA-M

InChI=1S/C21H20NO6.ClH/c1-22(2)6-5-11-7-15-16(26-9-25-15)8-13(11)18(22)19-12-3-4-14-20(27-10-24-14)17(12)21(23)28-19;/h3-4,7-8,18-19H,5-6,9-10H2,1-2H3;1H/q+1;/p-1/t18-,19+;/m0./s1

(6R)-6-[(5S)-6,6-dimethyl-7,8-dihydro-5H-[1,3]dioxolo[4,5-g]isoquinolin-6-ium-5-yl]-6H-furo[3,4-g][1,3]benzodioxol-8-one;chloride

bicuculline methochloride; 38641-83-7; (-)-BICUCULLINE METHOCHLORIDE; UNII-I3UNE1K4AF; N-Methylbicuculline; Bisculline methyl chloride; I3UNE1K4AF; Bicuculline (methochloride);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)