5HT1B receptor

Effects of the 5HT1B receptor agonist on [3H]-GABA release in the globus pallidus[1]
CP-93129 (5.4 μM) had no effect on basal [3H]-GABA release (P>0.05; n=8), but produced an approximate 45% reduction in [3H]-GABA release evoked at S2 (Figure 1b). Incubation with isamoltane (10 μM) similarly did not affect basal [3H]-GABA release (P>0.05; n=8) but reduced the inhibitory effects of CP-93129 on [3H]-GABA release evoked at S2 (Figure 1c).
The effects of increasing concentrations of CP-93129 on [3H]-GABA release ratios (S2/S1) are shown in Figure 2a (mean±s.e.mean; n=8). CP-93129 (0.6–16.2 μM) produced a significant concentration-dependent inhibition of 25 mM KCl-evoked [3H]-GABA release reaching a maximum inhibition of 52.5±4.5% with the highest concentration tested (16.2 μM) (see Figure 2a). Concentrations of CP-93129 below 0.6 μM failed to inhibit the release of [3H]-GABA (n=4; data not shown). Addition of isamoltane (10 μM) abolished the effect of 5.4 μM CP-93129 on [3H]-GABA release (P<0.05, n=8; Figure 2b).

Effects of intrapallidal 5HT1B receptor agonist administration in the reserpine-treated rat [1]
Post-mortem light microscopic examination confirmed the correct positioning of injection sites within the GP in approximately 80% of animals. Where aberrant injections were made adjacent to the GP, CP-93129 failed to induce any locomotor behaviour. Only data from those animals with correctly positioned cannulae were included in the analyses below.

During the baseline recordings, all reserpine-treated rats exhibited negligible locomotor activity (0 rotations 30 min−1) and were thus considered suitably akinetic for inclusion in the study. Unilateral injection of the 5HT1B receptor agonist, CP-93129 into the GP of reserpine-treated rats produced net contraversive rotations, the time-course for which is shown only for the maximum dose of CP-93129 (330 nmol) (Figure 3a). This rotational activity commenced immediately upon administration of CP-93129, reached a maximum rate of 10±2 turns 10 min−1 (n=8) and reversed back to baseline within 240 min. Subsequent quantification of locomotor activity induced by the full dose range of CP-93129 was thereafter made over 240 min (Figure 3b).

Neither vehicle nor low dose CP-93129 (30 nmol) produced any significant net contraversive rotations over this period. In contrast, CP-93129 (110–330 nmol) produced a dose-dependent increase in net contraversive rotations 240 min−1. Three of the eleven animals tested with the highest dose (330 nmol) were excluded from the analysis since they produced central excitation in the form of wet dog shakes and intermittent barrel rolling.

Pre-treatment with isamoltane (10 nmol) significantly inhibited the CP-93129 (220 nmol)-induced net contraversive rotations 240 min−1 by 84±6% (mean±s.e.mean, n=7), reflecting a decrease in both peak response and duration. Pre-treatment with vehicle for isamoltane (PBS, pH 7.4) did not affect the subsequent response to CP-93129 (n=6). No locomotor activity was observed during the equilibration period with isamoltane alone.

[3H]-GABA release studies [1]
The 5HT1B agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93129; Macor et al., 1990), (0.6, 1.8, 5.4 or 16.2 μM) or vehicle (aCSF) was included in the superfusate 16 min before, during and after S2 in order to examine its effects on release (n=8 animals per concentration). To confirm receptor specificity, isamoltane (10 μM), a relatively potent antagonist at 5HT1B receptors (Waldmeier et al., 1988), or vehicle (aCSF) was examined against a submaximal concentration of CP-93129 (5.4 μM) by inclusion in the superfusate 8 min prior to and during exposure to CP-93129 (n=8 animals per group).
Calcium dependency of the [3H]-GABA release was assessed in some slices by replacing the superfusate with Ca2+ free aCSF containing 5 mM EGTA between S1 and S2 (now 25 mM KCl in Ca2+ free aCSF).

Intrapallidal injections in reserpine-treated rats [1]
Under halothane anaesthesia, rats were stereotaxically implanted with 23 gauge stainless steel guide cannulae positioned 2 mm above the GP (co-ordinates: 0.92 mm posterior to and 3.0 mm lateral to bregma and 5.75 mm below the skull, according to the rat brain atlas of Paxinos & Watson, (1986)). Following a minimum of 5 days recovery, animals were treated with reserpine (5 mg kg−1, s.c.). Eighteen hours later, when animals displayed a stable level of akinesia, the effects of the 5HT1B receptor agonist, CP-93129, were assessed on motor behaviours.
Animals were placed in 40 cm diameter, flat-bottomed hemispheric bowls for visual assessment. Following a 20 min acclimatization period, baseline activity was videotaped for 30 min. Animals then received a single, unilateral injection of CP-93129 (30, 110, 220 or 330 nmol) in 0.5 μl phosphate-buffered saline (PBS) (mM: NaCl, 137; KCl, 2.7; KH2PO4 1.8; Na2HPO4, 10; pH 7.4) or vehicle (0.5 μl PBS) into the GP (n=8–11 animals per dose). Injections were made over a 2 min period via 30-gauge stainless steel needles inserted through, and extending 2 mm below the tip of the guide cannulae and attached with flexible (Portex) tubing to a 5 μl Hamilton microsyringe. Animals were videotaped for a further 330 min. Net contraversive rotations (360°C) were assessed as an index of unilateral relief of akinesia (Dawson et al., 2000). These rotations were counted manually from the videotape recordings in 10 min time bins. To confirm the receptor specificity of CP-93129, the effects of isamoltane (10 nmol) were examined against a single effective dose of CP-93129 (220 nmol). In these experiments, 7 h after the initial injection of CP-93129 (220 nmol), rats were injected with either isamoltane (10 nmol in 1 μl, pH 7.0; given over 5 min), or vehicle (1 μl PBS) into the same site (n=6–7 animals per group). Rotational behaviour was videotaped throughout the 30 min equilibration period for isamoltane and for a further 330 min following a repeat dose of CP-93129 (220 nmol). At the end of each experiment, fast blue dye (0.2 μl of 1%, w v−1) was injected via the guide cannulae to allow histological verification of injection sites. Approximately 5 min after dye injection, animals were killed by halothane overdose followed by cervical dislocation. The brains were rapidly frozen in isopentane (cooled to −45°C with solid CO2) and stored desiccated at −70°C until subsequent cryostat sectioning (20 μm) and cresyl violet (0.1% w v−1) staining.

[1]. The 5HT(1B) receptor agonist, CP-93129, inhibits [(3)H]-GABA release from rat globus pallidus slices and reverses akinesia following intrapallidal injection in the reserpine-treated rat. Br J Pharmacol. 2000 Aug;130(8):1927-32.

3-(1,2,3,6-tetrahydropyridin-4-yl)-1,4-dihydropyrrolo[3,2-b]pyridin-5-one is a pyrrolopyridine.

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This study examined whether activation of 5HT(1B) receptors in the rodent globus pallidus (GP) could reduce GABA release in vitro and reverse reserpine-induced akinesia in vivo. Microdissected slices of GP from male Sprague Dawley rats (300-350 g) were preloaded with [(3)H]-GABA. During subsequent superfusion, 4 min fractions were collected for analysis of release. The effects of the 5HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3, 2-b]pyrid-5-one (CP-93129), on 25 mM KCl-evoked release were examined using a standard dual stimulation paradigm. Male Sprague Dawley rats (270 - 290 g), stereotaxically cannulated above the GP, were rendered akinetic by injection of reserpine (5 mg kg(-1) s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of CP-93129 was examined. CP-93129 (0.6-16.2 microM) produced a concentration-dependent inhibition of 25 mM KCl-evoked [(3)H]-GABA release reaching a maximum inhibition of 52.5+/-4.5%. The effect of a submaximal concentration of CP-93129 (5.4 microM) was fully inhibited by the 5HT(1B) receptor antagonist, isamoltane (10 microM). Following intrapallidal injection, CP-93129 (30-330 nmol in 0.5 microl) produced a dose-dependent increase in net contraversive rotations reaching a maximum of 197+/-32 rotations in 240 min at 330 nmol. Pre-treatment with isamoltane (10 nmol in 1 microl) inhibited the effects of a submaximal dose of CP-93129 (220 nmol) by 84+/-6%. These data suggest that at least some 5HT(1B) receptor function as heteroreceptors in the GP, reducing the release of GABA. Moreover, CP-93129-mediated activation of these receptors in the GP provides relief of akinesia in the reserpine-treated rat model of PD.[1]

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This study examined the hypothesis that activation of 5HT1B receptors may reduce GABA release in the GP and thereby provide a way of alleviating parkinsonian akinesia. The data presented indicate that (i) 5HT1B receptor activation with CP-93129 does inhibit the release of [3H]-GABA from pallidal slices and (ii) intrapallidal injection of CP-93129 alleviates akinesia in the reserpine-treated rat model of PD. Taken together these data suggest a role for 5HT1B receptors in the regulation of GABAergic function in the GP that may prove useful in the search for novel therapeutic approaches for the treatment of PD.[1]

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In conclusion, these data indicate that some 5HT1B receptors can function as heteroreceptors in the GP, reducing the release of GABA from striatopallidal neurones. Moreover, this cellular mechanism may underlie the anti-akinetic activity of CP-93129 seen in the reserpine-treated rat model of PD.[1]

C12H15CL2N3O

288.17

287.059

C, 50.02; H, 5.25; Cl, 24.60; N, 14.58; O, 5.55

879089-64-2

879089-64-2 (2HCl); 127792-75-0

124007

Typically exists as solid at room temperature

3.578

3

2

1

16

361

0

C1CNCC=C1C2=CNC3=C2NC(=O)C=C3.Cl.Cl

PJYVGMRFPFNZCT-UHFFFAOYSA-N

InChI=1S/C12H13N3O/c16-11-2-1-10-12(15-11)9(7-14-10)8-3-5-13-6-4-8/h1-3,7,13-14H,4-6H2,(H,15,16)

3-(1,2,3,6-tetrahydropyridin-4-yl)-1,4-dihydropyrrolo[3,2-b]pyridin-5-one

CP 93129; CP 93129 DiHCl; CP 93129 DIHYDROCHLORIDE; 879089-64-2; CP-93129 Dihydrochloride Hydrate; 127792-75-0; 1,4-Dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrol[3,2-b]pyridin-5-one dihydrochloride; 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrol[3,2-b]pyridin-5-onedihydrochloride; 3-(1,2,3,6-Tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-b]pyridin-5(4H)-one dihydrochloride; cp93129 dihydrochloride; CP 93129 Dihydrochloride

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.)