IC50: 3 μM (GABAA)[3]

对 GABA 的最大响应在 1 μM 和 3 μM 的脉管碱甲基氯下实现。在表达人 α1β2γ2L GABAA 受体的非洲爪蟾卵母细胞中，氯丁胞苷似乎使 GABA 剂量反应曲线平行向右移动，而不降低最大 GABA 反应 [3]。这表明它是一个竞争性拮抗剂。在非洲爪蟾卵母细胞中，荷包牡丹碱甲基氯化物（1-100 μM；作为外用贴剂使用；2 分钟）可有效抑制 Apamin 不敏感的 K2 通道和 Apamin 敏感的小钙激活钾通道 (SK2) 电流。 SK1 中的敏感电流[4]。

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倍半萜三内酯白果内酯是50:1银杏叶提取物的活性成分之一，广泛用于增强记忆和学习。发现白果内酯可以拮抗γ-氨基丁酸（GABA）对重组α（1）β（2）γ（2L）GABA（A）受体的直接作用。采用双电极电压钳法评估了白果内酯对非洲爪蟾卵母细胞中表达的α（1）β（2）γ（2L）GABA（A）受体GABA直接作用的影响，并将其与经典的GABA（A）受体竞争性拮抗剂BicuuLline和非竞争性拮抗物苦味毒素的影响进行了比较。白果内酯（IC（50）=4.6+/-0.5微M）在α（1）β（2）γ（2L）GABA（A）受体上对40微M GABA（GABA EC（50））的效力几乎与荷包牡丹碱和苦托毒素（IC（50%）分别为2.0+/-0.1和2.4+/-0.5微m）一样强。虽然白果内酯和苦味毒素显然是非竞争性拮抗剂，但白果内酯的效力在高GABA浓度下会降低，这表明存在竞争性拮抗作用。[3]

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小电导钙激活钾通道（SK通道）仅由细胞内钙离子门控，其活性是许多可兴奋细胞中动作电位后缓慢后超极化（AHP）的原因。脑切片研究通常使用GABAA（γ-氨基丁酸）受体拮抗剂BicuuLine（bicuulline-m）的甲基衍生物来减少GABA能突触的强直性抑制作用，或研究这些突触在专门神经网络中的作用。然而，最近的证据表明，荷包牡丹碱-m可能对GABAA受体没有特异性，也可能阻断缓慢的AHP。因此，在爪蟾卵母细胞中表达后，研究了荷包牡丹碱-m对克隆的apamin敏感SK2和apamin不敏感SK1通道的影响。结果表明，在用于切片记录的浓度下，当应用于外向贴片时，荷包牡丹碱-m能有效阻断apamin敏感的SK2电流和apamin不敏感的SK1电流。Apamin不敏感的SK1电流在切除的斑块中下降。补丁切除后，荷包牡丹碱-m阻断的效力也随着时间的推移而降低。定点突变改变SK1孔外前庭中的两个残基，赋予apamin敏感性，也减少了贴片中电流的下降，并赋予了与SK2无法区分的荷包牡丹碱-m稳定的敏感性。因此，在切片记录中使用荷包牡丹碱-m可能会掩盖对apamin敏感的缓慢AHPs，而这些AHPs是神经元兴奋性的重要决定因素。此外，荷包牡丹碱-m不敏感的慢AHPs可能表明潜在的通道已经耗尽[4]。

在小鼠中，用巴布霉素氯甲烷（1.25-3 mg/kg；皮下注射）以剂量依赖性方式诱导阵挛性惊厥；给予吗啡（一种 μ-阿片受体激动剂）会加剧这些惊厥[1]。当皮下注射荷包牡丹碱氯甲烷（1.5-3.2 mg/kg）时，CD50（惊厥剂量）为 2.2 mg/kg（阵挛性）和 2.4 mg/kg（紧张性）的小鼠容易发生全身性癫痫发作。为了预防荷包牡丹碱引起的癫痫发作，可以静脉注射NMDA拮抗剂MK-801、CPP和CGS 19755作为预处理[2]。

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本研究调查了微量、δ和κ阿片受体激动剂对阻断γ-氨基丁酸（GABA）介导的小鼠突触传递产生的癫痫发作的影响。皮下注射选择性GABA（A）受体拮抗剂BicuuLine（1.25-3mg/kg）可引起剂量依赖性阵挛性强直性惊厥。在亚惊厥剂量的荷包牡丹碱前20分钟皮下注射原型μ阿片受体激动剂吗啡，可增强这些抽搐。用选择性μ阿片受体拮抗剂β-呋喃曲沙胺（0.5微克/只小鼠）进行脑室内预处理，吗啡的增强作用被完全逆转。用选择性δ阿片受体激动剂2-甲基-4aα-（3-羟基苯基）-1,2,3,4,4a，5,12,12aβ-八氢喹啉并[2,3,3-g]异喹啉（（（-）TAN-67）或[D-Pen（2,5）]-脑啡肽（DPDPE）进行心内预处理，显示抽搐发生率呈剂量依赖性增加。选择性δ阿片受体拮抗剂纳曲吲哚（2mg/kg，皮下注射）预处理可消除DPDPE对BicuuLine诱导的惊厥的增强作用。相比之下，选择性κ阿片受体激动剂U-50488H（0.6-80mg/kg，皮下注射或25-100微克/只小鼠，脑室注射）预处理对荷包牡丹碱诱导的惊厥产生了剂量依赖性的抑制作用。U-50488H的抑制作用完全被皮下注射选择性κ阿片受体拮抗剂诺比萘啡亚胺（5mg/kg）预处理所阻断。这项研究表明，μ阿片受体和δ阿片受体的激活会增加阻断GABA介导的突触传递引起的惊厥的发生率，而刺激κ阿片受体具有抗惊厥作用。[1]

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在小鼠中测试了兴奋性氨基酸拮抗剂对侧脑室（i.c.v.）或全身（s.c.）注射γ-氨基丁酸A（GABAA）拮抗剂荷包牡丹碱（BIC）诱导的惊厥的影响。3-[+/-）-2-羧基哌嗪-4-基）-1-丙基膦酸酯（CPP）、2-氨基-7-膦酰基庚酸酯（AP7）和（+）-5-甲基-10,11-二氢-5H-二苯并（a，d）环庚烷-5,10-亚胺马来酸酯（MK-801）用作N-甲基-d-天冬氨酸（NMDA）拮抗剂的代表。用作NMDA/KA混合拮抗剂。BicuuLine甲氧苄啶（BMI）静脉注射后，CD50为0.183 nmol（范围0.164-0.204），可诱导阵挛性惊厥。兴奋性氨基酸拮抗剂在侧脑室联合注射时，以0.224 nmol（约CD97）的剂量阻断BMI诱导的阵挛发作。CPP（ED50 0.0075 nmol）是最有效的抗惊厥药，其次是AP7（0.182 nmol）、MK-801（0.22 nmol），γ-d-GAMS（0.4 nmol）和KYNA（1.7 nmol）以及CNQX。（5.17 nmol）GABAA激动剂Muscimol（MSC）以0.25nmol的ED50阻断BMI诱导的癫痫发作。全身（皮下）注射BIC诱导小鼠全身性癫痫发作，阵挛的CD50为2.2 mg/kg（范围1.9-2.5），强直的CD50是2.4 mg/kg（范围2.2-2.7）。2+荷包牡丹碱在小鼠中引发癫痫发作的发病机制[2]。

电生理记录[3] 注射后2-8天，用双电极电压钳技术测量受体活性。记录微电极用微量移液器制造，并填充3 M KCl溶液。将卵母细胞置于细胞浴中，电压钳制在-60 mV。细胞持续用ND96缓冲液超灌。在Macintosh Quadra 605计算机上，使用Geneclamp 500放大器、Mac Lab 2e记录器和Chart 3.5.2版程序记录了药物应用引起的电流。测试了药物对GABAA受体GABA的直接激活。为了测量药物对受体激活的抑制作用，将药物加入含有GABA的缓冲溶液中，其浓度在受体处产生10%、50%、75%、90%和100%的效果（GABA EC10、EC50、EC75、EC90和EC100），以构建GABA抑制剂量-反应曲线。采用相同的程序，但拮抗剂浓度固定，GABA浓度增加，构建GABA剂量-反应曲线。每次用药之间允许3-5分钟的洗脱期，以防止受体脱敏。

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

63.22

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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