GABAA receptor; Bicuculline methiodide is a competitive antagonist of the GABAA receptor, binding to the same recognition site as the endogenous agonist GABA . Functional studies demonstrate that it completely blocks muscimol (a GABAA agonist)-stimulated 36Cl- uptake in brain microsomes, confirming its antagonistic activity on the receptor's chloride ion channel . The compound also exhibits off-target activity at small-conductance calcium-activated potassium (SK) channels and nicotinic acetylcholine receptors, though its primary research application remains as a GABAA antagonist .
(-)-Bicuculline methochloride is a potent and selective competitive antagonist of the GABA_A receptor. It binds to the GABA recognition site on the receptor, blocking the inhibitory effects of the endogenous agonist GABA. Antagonism can be overcome by increasing GABA concentration, demonstrating its competitive nature.

N-methyl-d-aspartate (NMDA) stimulation and burst firing of dopamine neurons are enhanced by cuculline methiodide (30 μM) [1]. According to studies, clustered LHb neurons in the tonic-firing, burst-firing, and silent categories are frequently found in the anti-reward central habenula (LHb) [2].

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Apamin, a bee venom toxin which blocks a Ca2+-dependent K+ current, potentiates N-methyl-D-aspartate (NMDA)-induced burst firing in dopamine neurons. We now report that burst firing is also potentiated by an apamin-like effect of bicuculline methiodide (BMI) at the same concentration (30 microM) which blocks GABA(A) receptors in vitro. Using microelectrodes to record intracellularly from rat dopamine neurons in the midbrain slice, BMI reduced the apamin-sensitive afterhyperpolarization in all cells tested. BMI also mimicked apamin (100 nM) by potentiating burst firing produced by a concentration of NMDA (10 microM) which is too low to evoke burst firing when perfused alone. When recording under voltage-clamp, both BMI and apamin reduced a depolarization-activated outward current which was also sensitive to perfusate containing no-added Ca2+. Although picrotoxin (100 microM) and bicuculline free base (30 microM) blocked the inhibition of firing produced by the GABA(A) agonist isoguvacine (100 microM), neither had apamin-like effects. We conclude that BMI potentiates burst firing by blocking an apamin-sensitive Ca2+-activated K+ current [1].

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Bicuculline methiodide acts as a potent GABAA receptor antagonist in vitro. In standard radioligand binding assays using rat brain synaptic membranes, it effectively displaces GABA from the agonist binding site, preventing receptor activation, and also acts as a negative allosteric inhibitor of channel opening . Moreover, it can completely block muscimol-stimulated 36Cl- uptake in brain microsomes, an effect that is dose-dependent. Interestingly, the compound displays region-specific effects in the brain; for instance, it enhances 35S-TBPS binding to the convulsant site on the GABAA receptor complex in the cortex and cerebellum, while inhibiting it in the inferior colliculus, indicating regional heterogeneity of the GABAA receptor complex .

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In radioligand binding assays using rat brain synaptosomal membranes, (-)-bicuculline methochloride decelerates the kinetics of [³H]TBOB (a convulsant site ligand) binding in a manner opposite to that of GABA, reflecting its antagonistic effect on the GABA_A receptor complex. It also reverses the inhibitory effect of GABA on [³H]TBOB binding completely, a property that is predictive of a compound‘s proconvulsive potential. Furthermore, in electrophysiological studies using acutely dissociated rat hippocampal neurons, bicuculline methochloride acts as a competitive antagonist, reducing the sensitivity of the GABA_A receptor to GABA.

The in vivo effects of bicuculline methiodide are highly dependent on the route of administration due to its poor blood-brain barrier permeability. When microinjected directly into brain regions such as the basolateral amygdala (BLA), it acts as a potent anxiogenic and cardiovascular stressor, increasing heart rate, blood pressure, and anxiety-like behavior (measured by decreased social interaction time) in rats, which are effects that can be blocked by co-administration of NMDA (e.g., AP5, dizocilpine) or non-NMDA (e.g., CNQX) glutamate receptor antagonists . When administered systemically (e.g., intraperitoneally) at doses as high as 36 mg/kg, it typically produces no consistent behavioral or EEG abnormalities in rats because it cannot effectively cross the BBB. However, this changes dramatically in the presence of a focal BBB lesion, where the same dose can induce intense, highly localized epileptiform discharges, highlighting the compound's utility in the BBB-epileptogen model of focal epilepsy .

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The effects of (-)-bicuculline methochloride are highly dose- and route-dependent. Local infusion into the mouse somatosensory cortex induces focal interictal (epileptiform) activity and significantly increases local capillary blood flow within 300 µm of the infusion site, providing a model for studying the hemodynamics of epileptic foci. Local application to the canine ventral respiratory group produces a marked, dose-dependent increase in neuronal discharge frequency, revealing a tonic GABAergic gain modulation that normally suppresses respiratory patterns. Systemic administration in rats induces clonic-tonic convulsions, making it a valuable tool for studying seizure mechanisms and screening anticonvulsant drugs at doses of 1-4 mg/kg.

A standard cell-free method to study the GABAA receptor is the radioligand binding assay using 3H-(+)-bicuculline methiodide itself. In this approach, synaptic membrane preparations from rat CNS tissue are incubated with the radiolabeled antagonist. Historically, this was considered more suitable for receptor studies than using 3H-GABA because bicuculline, unlike GABA, does not bind to glial or neuronal uptake sites nor to GABA-metabolizing enzymes, offering a more selective profile for identifying the receptor .

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A classic cell-free protocol to study the GABA_A receptor is a radioligand binding assay using [³H]TBOB, a convulsant site ligand. Rat brain synaptosomal 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 initiated by adding the membrane preparation and terminated by rapid filtration through glass fiber filters. The amount of bound [³H]TBOB is determined by liquid scintillation counting. This assay can demonstrate the antagonistic effect of bicuculline, which decelerates the dissociation rate (off-rate) of [³H]TBOB, contrary to the effect of GABA.

Specific in vitro cell assay protocols for bicuculline methiodide often utilize electrophysiological techniques. In a classic setup for studying synaptic transmission, researchers can isolate glutamate receptor-mediated excitatory postsynaptic currents in brain slices. The protocol involves perfusing the slice with artificial cerebrospinal fluid containing bicuculline methiodide (typically at concentrations around 10-30 µM) to completely block GABAA-mediated inhibitory postsynaptic currents, thereby allowing for the selective recording of glutamatergic events. This approach is widely used because bicuculline methiodide is water-soluble and effective at this common concentration . For functional studies, as described in the literature, 36Cl- uptake assays in brain microsomes are also employed to measure chloride channel function. Following the addition of GABA, muscimol (a GABAA agonist), and the test compound, the amount of radiolabeled chloride entering the vesicles is quantified . In some cases, the compound has been used in invertebrate models; for example, in C. elegans, it has helped demonstrate pharmacological distinctions between UNC-49 GABA receptors and mammalian GABAA receptors .

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Cell-based assays for (-)-bicuculline methochloride typically use the whole-cell patch-clamp technique on acutely dissociated neurons or brain slices. For instance, to study synaptic transmission, a brain slice is continuously perfused with an artificial cerebrospinal fluid (ACSF) containing a low concentration (e.g., 0.01 mM) of bicuculline methochloride to specifically block GABA_A-mediated inhibitory postsynaptic currents (IPSCs). This allows for the isolation and recording of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by stimulating specific afferent pathways, such as parallel fibers in the cerebellum.

In a widely cited animal protocol for studying the BLA's role in anxiety, a method is described where researchers use adult male Wistar rats. On the day of the experiment, a microinjection cannula is inserted bilaterally into the BLA. Bicuculline methiodide is dissolved in aCSF, and a precise volume (e.g., 100 nL) containing a low dose (e.g., 20 pmol, not 20 nmol) is infused into the brain region over a set time (e.g., 60 sec). The behavioral effects are then quantified using the social interaction test, while physiological changes (increases in heart rate and blood pressure) are simultaneously monitored via an arterial catheter. This low picomolar dose is highly effective when injected directly into the brain but would be ineffective if given systemically .

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A well-established protocol to study focal epilepsy involves anesthetizing a mouse (e.g., with ketamine-xylazine) and performing a craniotomy to expose the somatosensory cortex. A glass micropipette is inserted into the cortex, and (-)-bicuculline methochloride is infused locally (e.g., 100 µM) to create an epileptic focus. Two-photon laser scanning microscopy can then be used to quantify red blood cell flux in cortical capillaries as a measure of local cerebral blood flow, which has been shown to increase significantly near the focus. For systemic seizure induction, rats are administered bicuculline methochloride intravenously or intraperitoneally at doses of 1-4 mg/kg to induce clonic-tonic convulsions.

The pharmacokinetics of bicuculline methiodide are defined by its poor ability to cross the BBB due to its permanently charged quaternary ammonium group. In a rat model, systemic administration at a high dose of 36 mg/kg produces no overt CNS effects, indicating that brain penetration is minimal under normal conditions . However, it can be distributed into the brain effectively if the BBB is compromised. It is soluble in water and DMSO, which facilitates its use in both patch-clamp electrophysiology and in vivo microinjection experiments .

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(-)-Bicuculline methochloride is a quaternary ammonium compound, a property that gives it very poor bioavailability in the central nervous system after peripheral administration due to its inability to passively cross the blood-brain barrier. It is primarily designed for direct, local administration into the brain (e.g., by microinjection or microiontophoresis) to achieve its effects. The compound is soluble in water, which makes it suitable for these application methods.

Bicuculline methiodide exhibits low systemic toxicity due to its poor bioavailability in the CNS. At a dose of 36 mg/kg administered intraperitoneally in rats, no consistent abnormalities in behavior or EEG are observed because it cannot effectively reach its central target . This is in stark contrast to its parent compound, bicuculline, which crosses the BBB easily and is highly toxic, inducing severe seizures at much lower doses. The safety profile is enhanced because the compound remains primarily in the peripheral circulation.

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(-)-Bicuculline methochloride is a potent central nervous system convulsant. Its primary toxicity is a direct consequence of its on-target pharmacology as a GABA_A antagonist. Systemic administration at doses as low as 1-4 mg/kg can induce clonic-tonic convulsions and status epilepticus in animal models. This proconvulsant effect is dose-dependent, with higher doses leading to more severe and rapid-onset seizures. It is a standard tool in seizure research specifically because of this well-characterized toxicity.

[1]. Bicuculline methiodide potentiates NMDA-dependent burst firing in rat dopamine neurons by blocking apamin-sensitive Ca2+-activated K+ currents. Neurosci Lett. 1997 Aug 1;231(1):13-6.

[2]. Ketamine blocks bursting in the lateral habenula to rapidly relieve depression. Nature, 2018, 554(7692): 317-322.

Bicuculline methiodide (CAS: 40709-69-1) is a yellow solid that is soluble in water (at least 10 mg/mL) and DMSO (up to 25 mg/mL) . It is often stored at room temperature or 2-8°C . While it is a derivative of the light-sensitive alkaloid bicuculline, the methiodide version is significantly more stable, making it more practical for laboratory use .

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(-)-Bicuculline methochloride is a synthetic, water-soluble, and more stable alternative to the parent compound (+)-bicuculline. It has a molecular weight of 417.85 g/mol. In research, it has been instrumental in identifying a novel GABAergic gain modulation in the brainstem that multiplies neuronal output patterns, an effect that is mechanistically distinct from the blockade of small-conductance calcium-activated potassium (SK) channels. Its use continues to be critical in understanding the role of inhibition in cortical processing, epilepsy, and synaptic physiology.

C21H20INO6

509.2911

509.034

C, 49.53; H, 3.96; I, 24.92; N, 2.75; O, 18.85

40709-69-1

(-)-Bicuculline methobromide;73604-30-5;Bicuculline;485-49-4;(-)-Bicuculline methochloride;53552-05-9;Bicuculline methobromide;66016-70-4;Bicuculline methochloride;38641-83-7

104871

White to off-white solid powder

0

7

1

29

655

2

C[N+]1(CCC2=CC3=C(C=C2[C@H]1[C@H]4C5=C(C6=C(C=C5)OCO6)C(=O)O4)OCO3)C.[I-]

HKJKCPKPSSVUHY-VOMIJIAVSA-M

InChI=1S/C21H20NO6.HI/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

1,3-Dioxolo(4,5-g)isoquinolinium, 5-(6,8-dihydro-8-oxofuro(3,4-e)-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-, iodide, (R-(R*,S*))-

d-Bicuculline; Bicculine; Bicucullin; BRN 0098786; 40709-69-1; Bicuculline methiodide; (-)-BICUCULLINE METHIODIDE; 1(S),9(R)-(-)-Bicuculline methiodide; MLS000069548; MFCD00078966; (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;iodide; [R-(R*,S*)]-5-(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 iodide; NSC 32192; BRN0098786; NSC32192; BRN-0098786; NSC-32192

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~150 mg/mL (~294.53 mM)

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.)