Glycine receptor

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (alpha1beta1gammadelta, alpha1beta1gamma, and alpha1beta1delta) and neuronal (alpha2beta2 and alpha2beta4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 microM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was alpha1beta1gammadelta > alpha2beta4 > alpha2beta2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of approximately 0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex[1].

Strychnine-sensitive glycine receptors are expressed in many adult forebrain regions, yet the biological function of these receptors outside the spinal cord/brainstem is poorly understood. We have recently shown that rat lateral/basolateral amygdala neurons express strychnine-sensitive glycine-gated currents whose pharmacological and molecular characteristics are consistent with those established for classic ligand-gated chloride channels. The current studies were undertaken to establish the behavioral role, if any, of these strychnine-sensitive glycine receptors. Adult Long-Evans male rats were implanted with guide cannulae targeted at the lateral amygdala and were microinjected with standard artificial cerebrospinal fluid with or without various doses of strychnine or taurine. Anxiety-like behaviors were assessed with the elevated plus maze or the light/dark box. In the elevated plus maze, strychnine decreased closed-arm time and increased open-arm time, suggestive of an anxiolytic effect. Similarly, strychnine produced a modest anxiolytic effect in the light/dark box. Post hoc analysis of 'open-arm' time and 'light-side' time indicated that aCSF-treated animals were distributed into two apparent groups that displayed either high or low amounts of anxiety-like behavior in a given apparatus. Surprisingly, the pharmacological effects of both strychnine and taurine in these assays were dependent upon a given animal's behavioral phenotype. Together, these findings are significant because they suggest that the basal 'emotional state' of the animal could influence the behavioral outcome associated with drug application directly into the lateral/basolateral amygdala. Furthermore, our findings also suggest that compounds acting at amygdala strychnine-sensitive glycine receptors may actively modulate this basal anxiety-like state[2].

Membrane currents were recorded, at room temperature (20–23°C), 3–9 days after cRNA injection by using a voltage-clamp technique with two microelectrodes filled with 3 M KCl. The oocytes were continuously superfused in a recording chamber (volume ≈ 0.1 ml) at a rate of 7–10 ml/min with normal frog Ringer’s solution (in mM): 115 NaCl, 2 KCl, 1.8 CaCl2, 5 Hepes adjusted with NaOH, at pH 7.0. Ionic currents were recorded with a digital oscilloscope and were stored in discs for subsequent analyses by using a program made by Rico Miledi. AcCho and strychnine were diluted daily in normal Ringer from concentrated frozen stocks and were applied via the superfusion system[1].

Animals were randomly assigned to different treatment groups and were bilaterally micro-injected with standard artificial cerebrospinal fluid (aCSF) with or without various doses of strychnine or taurine. Each week, animals were exposed to a single injection followed by testing in a single apparatus. To reduce the ‘one-trial tolerance’ effect [4] in the various assays, animals exposed to one apparatus on any given week were tested on another apparatus the following week; animals were exposed to any given apparatus only once every three weeks. A 2×2 or 3×3 Latin-square design was used to assign subjects to an aCSF-injected and strychnine- or taurine-injected groups. For example, during the strychnine studies, animals were divided into two equivalent groups; each group was injected with either aCSF or strychnine (0.5pmol or 167pg) and subsequently exposed to a single behavioral apparatus on the first week of testing. On the second week, aCSF and strychnine ‘groups’ were switched, injected, and tested on a separate apparatus. This process was repeated until each animal had received both aCSF and drug injections and had been tested on each apparatus. Taurine experiments were divided into two studies, for the first study, each animal received an aCSF, 50pmol taurine (6.3ng), or 1nmol (125ng) taurine microinjection. In the second study, each animal received aCSF or 5pmol (0.6ng) taurine. Again, injection order and exposure to each apparatus was counterbalanced across weeks such that animals receiving one type of injection and tested on a given apparatus one week would receive a different drug and behavioral assay the next week. We and others have previously shown that repeated microinjections into the same brain region does not substantially influence drug sensitivity even over several administrations (see [33, 42, 44]). For all dependent variables examined, the values of aCSF-injected animals across the two experiments did not differ; and these groups were combined for further analysis. For both the strychnine and taurine studies, there was a significant effect of previous apparatus exposure on the number of vertical-plane entries (rears) and the ‘egress latency’ in the light/dark box. These behaviors were therefore excluded from analysis. There were no trial-dependent effects for any plus maze dependent variable[2].

[1]. Modulation of nicotinic acetylcholine receptors by strychnine. Proc Natl Acad Sci U S A . 1999 Mar 30;96(7):4113-8.
[2]. Strychnine and taurine modulation of amygdala-associated anxiety-like behavior is 'state' dependent. Behav Brain Res . 2007 Mar 12;178(1):70-81.

Strychnine hydrochloride is a hydrochloride salt prepared by reacting strychnine with an equimolar amount of hydrogen chloride. It possesses effects as a bird antagonist, cholinergic antagonist, glycine receptor antagonist, rodenticide, and neurotransmitter. It is a hydrochloride and an organic ammonium salt containing strychnine (1+) ions.

C21H23CLN2O2

370.87

370.144

C, 68.01; H, 6.25; Cl, 9.56; N, 7.55; O, 8.63

1421-86-9

1421-86-9 (HCl);57-24-9;60-41-3 (sulfate);66-32-0 (nitrate);

16219987

Typically exists as solid at room temperature

559.9ºC at 760mmHg

295 °C

292.4ºC

1.44E-12mmHg at 25°C

2.897

1

3

0

26

689

6

C1=CC=C2C(=C1)[C@]34CCN5CC6=CCO[C@H]7CC(=O)N2[C@H]4[C@H]7[C@H]6C[C@@H]35.Cl

VLXYTKMPCOQKEM-ZEYGOCRCSA-N

InChI=1S/C21H22N2O2.ClH/c24-18-10-16-19-13-9-17-21(6-7-22(17)11-12(13)5-8-25-16)14-3-1-2-4-15(14)23(18)20(19)21;/h1-5,13,16-17,19-20H,6-11H2;1H/t13-,16-,17-,19-,20-,21+;/m0./s1

(4aR,4a1R,5aS,8aR,8a1S,15aS)-2,4a,4a1,5,5a,7,8,8a1,15,15a-decahydro-14H-4,6-methanoindolo[3,2,1-ij]oxepino[2,3,4-de]pyrrolo[2,3-h]quinolin-14-one hydrochloride

Strychnine HCl; Strychnine hydrochloride; 1421-86-9; STRYCHNINE HCl; Strychnine, monohydrochloride; Strychnidin-10-one, hydrochloride (1:1); Strychnidin-10-one, monohydrochloride; CHEBI:90699; strychnidin-10-one hydrochloride

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.)