GABAA receptor

Bicuculline itself is difficult to use because it is unstable at physiological pH (Ref. 8) and not very soluble in aqueous solutions. This can be a problem, particularly for iontophoresis and superfusion of in vitro preparations and so, in response to this, the quaternary N-methyl derivative was synthesized and various salts (bicuculline methiodide, methochloride and methobromide, which we will refer to as BIC salts) were made available. The base of bicuculline and BIC salts was used extensively as specific GABAA receptor antagonists. It should be noted that recent experiments using recombinant receptors have modified the classical view of the interaction of bicuculline and SR95531 with the GABAA receptor. Indeed, it has been suggested that both compounds might have a negative allosteric effect (i.e. be inverse agonists) rather than (or in addition to) being competitive antagonists.
Several reports have shown that bicuculline and BIC salts also affect other neurotransmitter systems (e.g. the cholinergic system) and possibly ion channels, but results from these studies, which are summarized in Table 1, have been overlooked by many investigators. There appear to be two main reasons for this. First, actions on the cholinergic system should be easily distinguished from the GABAergic effect (for example, block of nicotinic responses will have an inhibitory effect instead of the classical excitatory effect of bicuculline) and second, some of the drugs' effects (e.g. effects on spinal cord neurones) occurred at rather high concentrations [1].

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(-)-Bicuculline methochloride is a potent and selective competitive antagonist of the GABAA receptor, binding to the GABA recognition site on the receptor-channel complex . Its antagonism is surmountable by increasing GABA concentration, confirming its competitive nature .

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(-)-Bicuculline methochloride potently blocks GABA-evoked currents in heterologous expression systems, with effects completely reversible upon washout . In radioligand binding assays using rat brain synaptosomal membranes, the compound decelerates the kinetics of convulsant site ligand binding, an effect opposite to that of GABA, reflecting its antagonistic action on the receptor complex .

(-)-Bicuculline memethochide (0.6 nmol/rat) attenuates Neurotropin's anti-allodynic effect [2]. IV (50-100 NU/kg) doses of Neurotropin elicited an antiallodynic action in L5-SNL rats. Moreover, intracerebroventricular (400 mNU/rat), but not intrathecal, injection of Neurotropin inhibited allodynia. The antiallodynic action of Neurotropin (100 NU/kg, IV) was antagonized by intrathecal injections of yohimbine (10 nmol/rat), ketanserin (30 nmol/rat), MDL72,222(30 nmol/rat), bicuculline (0.6 nmol/rat) and CGP35348 (30 nmol/rat). On the other hand, the antiallodynic action of intrathecally injected m-CPBG (5-HT(3) receptor agonist) was reversed by intrathecal injection of bicuculline and CGP35348, suggesting interaction of 5-HT(3) receptors and spinal inhibitory (GABAergic) interneurons. Conclusions: These results suggest that the antiallodynic effect of Neurotropin is mediated via activation of descending pain inhibitory systems, such as the noradrenergic and serotonergic systems, which project from supraspinal sites to the spinal dorsal horn. In addition, activation of inhibitory GABAergic interneurons via 5-HT(3) receptors by serotonin released in the spinal dorsal horn may also be involved in the antiallodynic action of Neurotropin [2].

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The effects of (-)-bicuculline methochloride are highly dose- and route-dependent. When applied locally in vivo via microiontophoresis, it selectively blocks GABA- but not glycine-induced responses in rat brainstem neurons . In hippocampal slice cultures, prolonged exposure (48 hr) to 100 μM (-)-bicuculline methochloride induces CA1 pyramidal cell death, making it a reliable in vitro model for epileptiform insult-induced neurodegeneration .

A classic cell‑free protocol for studying the GABAA receptor involves a [³H]TBOB binding assay using rat brain synaptosomal membranes. Membranes are prepared and incubated with a fixed concentration of [³H]TBOB. To assess modulation, (-)-bicuculline methochloride (1 µM) is added to the assay mixture. The reaction is terminated by rapid filtration through glass fiber filters, and bound radioactivity is quantified by liquid scintillation counting. This assay demonstrates that bicuculline methochloride reverses the inhibitory effect of GABA on [³H]TBOB binding, a property that predicts proconvulsive potential .

Block of Ca2+-activated K+ currents by BIC salts (bicuculline methiodide, methochloride and methobromide) in mammalian neurones. These experiments were performed on brain slices using either intracellular recordings (a, b) or whole-cell patch-clamp recordings (c). a: Concentration-dependent blockade of the apamin-sensitive afterhyperpolarization (AHP) by a BIC salt in a mesencephalic dopaminergic neurone. Note that the maximal effect of bicuculline methochloride (BMC) is similar to the one of a maximally active concentration of apamin. b: Comparison of the potency of BMC as a GABAA receptor antagonist (circles) and as a blocker of the AHP (squares) in dopaminergic neurones. The antagonism at GABAA receptors was estimated by the ability of the BIC salts to antagonize the increase in conductance induced by muscimol (3 μm) (not shown). Note that the two curves partially overlap. c: Concentration-dependent enhancement by a BIC salt of the rebound low-threshold spike in a thalamic reticular nucleus neurone. This effect is due to a block of the apamin-sensitive current. Note that it occurs at concentrations similar to the ones that block this current in dopaminergic neurones [1].

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For cell‑based assays, the whole‑cell patch‑clamp technique is commonly employed. Cells (e.g., Xenopus oocytes expressing recombinant GABAA receptors) are voltage‑clamped at a holding potential (e.g., -60 mV). GABA is applied via fast perfusion to evoke inward currents, and the reduction in current amplitude upon co‑application with (-)-bicuculline methochloride is measured. The competitive nature of the antagonism can be confirmed by constructing GABA concentration‑response curves in the presence and absence of a fixed concentration of the antagonist .

Animal/Disease Models: Rat L5-SNL model [2]
Doses: 0.6 nmol/rat
Route of Administration: Intrathecal injection (100 NU/kg, intravenous (iv) (iv)injection) 5 minutes before administering Neurotropin
Experimental Results:Attenuated the anti-allodynic effect of Neurotropin .

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The left fifth lumbar nerve of rats was tightly ligated with silk sutures under pentobarbital anesthesia. Mechanical allodynia was confirmed by measuring the hindpaw withdrawal threshold in response to application of von Frey filaments. Behavioral tests were performed at 28 days after nerve ligation. Neurotropin was administered IV, intrathecally or intracerebroventricularly in L5 spinal nerve ligation (L5-SNL) rats. We examined the effects of noradrenergic, serotonergic and gamma-aminobutyric acid (GABA)ergic antagonists on the antiallodynic action of Neurotropin in L5-SNL rats. Yohimbine hydrochloride (yohimbine) was used as an alpha(2) adrenoceptor antagonist, ketanserin tartrate (ketanserin) as a 5-HT(2A) receptor antagonist, MDL72,222 as a 5-HT(3) receptor antagonist, (-)-bicuculline methobromide (bicuculline) as a GABA(A) receptor antagonist, and CGP35,348 as a GABA(B) receptor antagonist, and intrathecally injected. [2]

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A widely used protocol to study the antagonism of GABAergic responses in vivo involves microiontophoresis in anesthetized rats. A multibarrel micropipette is positioned in a brain region such as the cuneate nucleus. One barrel is filled with (-)-bicuculline methochloride (5 mM), and adjacent barrels contain GABA and glycine for agonist application. Ejection currents (up to 100 nA) are used to apply the antagonist, and neuronal firing rates are recorded extracellularly. The ability of the antagonist to selectively block GABA- over glycine-induced inhibition is then assessed .

(-)-bicuculline methochloride is known to be water-soluble, which facilitates its use in microinjection and microiontophoresis experiments. Its quaternary ammonium structure gives it a fixed positive charge, which restricts its ability to passively diffuse across lipid membranes, including the blood-brain barrier, after systemic administration .

(-)-Bicuculline methochloride is a potent neurotoxin classified as acute toxic category 1 (oral), with the hazard statement “Fatal if swallowed” (H300) . It is also listed as toxic in contact with skin (H311) and toxic if inhaled (H331). The compound is very toxic to aquatic life (H400) and must not be released into the environment . In ex vivo hippocampal slice cultures, exposure to 100 µM for 48 hours is sufficient to induce selective CA1 pyramidal cell death, highlighting its neurotoxic potential at higher concentrations or prolonged exposure times .

[1]. Recent advances in the pharmacology of quaternary salts of bicuculline. Trends Pharmacol Sci. 1999 Jul;20(7):268-70.

[2]. The antiallodynic effect of Neurotropin is mediated via activation of descending pain inhibitory systems in rats with spinal nerve ligation. Anesth Analg. 2008 Sep;107(3):1064-9.

Neurotropin is a non-protein extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus. It is widely used in Japan to treat chronic pain, such as neuropathic pain. Although some studies have explored the mechanism of neurotropin's anti-atopic pain action, the mechanism has not been fully elucidated. [2]

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(-)-Bicuculline methochloride (CAS: 53552-05-9) has a molecular weight of 417.85 and a purity of ≥99% by HPLC for research-grade compounds . While widely used as a GABAA antagonist, it is important to note that at higher concentrations (>5 µM), it can directly block small-conductance calcium-activated potassium (SK) channels, an off-target effect that should be considered when interpreting results . For electrophysiological studies requiring GABAA blockade without SK channel involvement, picrotoxin or bicuculline free base are recommended alternatives .

C₂₁H₂₀CLNO₆

417.8396

417.098

53552-05-9

(-)-Bicuculline methobromide;73604-30-5;Bicuculline methiodide;40709-69-1;Bicuculline;485-49-4

56972147

Typically exists as solid at room temperature

0

7

1

29

655

2

C[N+]1(CCC2=CC3=C(C=C2C1C4C5=C(C6=C(C=C5)OCO6)C(=O)O4)OCO3)C.[Cl-]

RLJKFAMYSYWMND-VOMIJIAVSA-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,5-6,9-10H2,1-2H31H/q+1/p-1/t18-,19+/m1./s1

(5R)-5-[(6S)-6,8-dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl]-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium, monochloride

l-Bicuculline methochloride; 53552-05-9; (-)-BICUCULLINE METHOCHLORIDE; (-)-Bicuculline (methochloride); (?)-Bicuculline methochloride; (6S)-6-[(5R)-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

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.)