In gramicidin perforated experiments and in experiments performed for assessing the KCC2 efficacy, GABAAR responses were isolated by using a cocktail of drugs containing 0.2 μM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 μM (+)-tubocurarine (Millipore Sigma), 5 μM Dihydro-β-erythroidine hydrobromide (DHβE, Bio-techne, France), and 3 μm strychnine (Millipore Sigma) that respectively blocked voltage-dependent Na+ action potentials, and glutamate, cholinergic, and glycinergic input to MNs. In CsCl experiments, IPSCs were isolated pharmacologically using DL-AP5 40 μM ((2R)-amino-5-phosphonovaleric acid, Bio-techne, France) and CNQX 20 μM (6-cyano-7-nitroquinoxaline-2,3-dione, Bio-techne, France). mIPSCs were isolated in the presence of 0.2 μM TTX (Latoxan, France). GABA and glycine mIPSCs were isolated by adding 3 μM strychnine or 3 μM GABAzine (SR 95531 hydrobromide, Bio-techne, France), respectively. These blockers were added to the cocktail containing 0.2 µM TTX, 4 mM kynurenic acid, 10 μM (+)-tubocurarine and 5 μM DHβE. In experiments assessing the KCC2 efficacy, 10 µM bumetanide (Millipore Sigma) was applied to block NKCC1 and 10 µM VU0240551 (Bio-techne, France) (10 µM) to block KCC2. Serotonin (5-HT, 10 µM), dopamine (DA, 100 µM) and N-Methyl-D-aspartic acid (NMDA, 10 µM) were from Millipore Sigma.
Serotonin 5 ht
Manufactured by Merck Group
Sourced in United States
Serotonin (5-HT) is a neurotransmitter that plays a crucial role in various physiological processes. It is a monoamine compound found primarily in the gastrointestinal tract, blood platelets, and the central nervous system. Serotonin is involved in the regulation of mood, sleep, appetite, and other important functions.
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Lab products found in correlation
Strychnine, by Merck Group (2 mentions)
Kynurenic acid, by Merck Group (2 mentions)
Tubocurarine, by Merck Group (2 mentions)
Vu0240551, by Bio-Techne (2 mentions)
Dihydro β erythroidine hydrobromide dhβe, by Bio-Techne (2 mentions)
Cysteamine, by Merck Group (2 mentions)
Ru486, by Merck Group (2 mentions)
Fluoxetine, by Merck Group (2 mentions)
Corticosterone, by Merck Group (2 mentions)
Hydroxypropyl β cyclodextrin, by Merck Group (2 mentions)
Corticosterone 4 pregnen 11b diol 3 20 dione 21 hemisuccinate, by Merck Group (2 mentions)
Brain derived neurotrophic factor (bdnf), by ProSpec (2 mentions)
U46619, by Merck Group (2 mentions)
Wire myograph, by Danish Myo Technology (2 mentions)
N methyl d aspartic acid, by Merck Group (1 mentions)
Male c57bl 6 mice, by Japan SLC (1 mentions)
Bat7001h, by Physitemp (1 mentions)
5 hydroxytryptophan 5 htp, by Merck Group (1 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
5 biotinamidopentylamine 5 bp, by Thermo Fisher Scientific (1 mentions)
23 protocols using serotonin 5 ht
1
Isolating GABAAR Responses for Functional Analysis
2
Pharmacological Isolation of GABAAR Responses
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In the gramicidin perforation experiments, GABAAR responses were isolated by using a cocktail of drugs containing 0.2 µM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 µM (+)-tubocurarine (Millipore Sigma), 5 µM dihydro-β-erythroidine hydrobromide (DHΒE, Bio-Techne, Lille, France), and 3 µM strychnine (Millipore Sigma), which blocked voltage-dependent Na+ action potentials and glutamate, cholinergic, and glycinergic inputs to MNs, respectively. VU0240551 (Bio-Techne, France) (10 µM) was applied to specifically block KCC2. Serotonin (5-HT, 10 µM) was purchased from Millipore Sigma. Methysergide maleate (10 µM) and ketanserin tartrate (10 µM) (Bio-techne) were used to evaluate the involvement of 5-HTRs in the effect of 5-HT. Combined, these antagonists are known to efficiently block 5-HT2A-CR [31 (link),32 (link)].
3
Corticosterone-Induced Neuronal Responses
4
Serotonin Effects on Metabolic Regulation in Mice
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Male C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). All mice were housed in a temperature-controlled facility (23°C) with a 12-hour light/dark cycle and fed a chow diet (14.4 MJ/kg) containing 4.8% fat (Ch) or high-fat diet (17.0 MJ/kg) containing 13.6% fat (F) (CLEA Japan, Inc., Tokyo, Japan). The mice were injected i.p. with serotonin (5-HT) (0.1 mg, 0.5 mg or 1 mg) (Sigma, St. Louis, MO) or phosphate buffered saline (PBS) twice a week between the ages of 5 and 26 weeks. The body weight of mice was measured at the same time as the injections were given. The mice were fasted 12 h before blood and tissues samples were harvested in all experiments. The food intake in each group mice was measured for 5 days when they were 17 weeks of age. The rectal temperature was measured with a thermometer (BAT-7001H; Physitemp Instruments Inc, Clifton, NJ) at 26 weeks of age. The experiments were permitted by the Tohoku University Environmental & Safety Committee and conducted in accordance with the Guidelines for Animal Experimentation of Tohoku University, which have been sanctioned by the relevant committee of the Government of Japan.
5
Serotonin Effects on Developmental Stages
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D. vorticoides at different developmental stages were incubated in 10−5 M serotonin (5-HT) and 5-hydroxytryptophan (5-HTP) (Sigma–Aldrich, Burlington, MA, USA) for 3 h. Fresh stock solutions in distilled water (10−2 M) were prepared immediately before each experiment. Negative controls for each experiment included treatment of animals with the final concentration of vehicle (distilled water) in filtered seawater. The experiments were performed at 10 °C. After the incubation, the animals were washed with filtered seawater four times and were then fixed and stained with anti-5-HT and anti-tubulin antibodies as described above.
6
Serotonin Receptor Modulator Protocols
7
Corticosterone-Induced Neuronal Responses
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NSC 23766 (NSC, Tocris) and Fluoxetine (Sigma, St Louis, MO, USA) were administrated intraperitoneally (i.p; 100 μL) at a concentration of 2.5 mg per kg body weight per day (mg kg-1 day-1) and 20 mg kg-1 day-1 respectively. Cysteamine (Sigma) was administrated at a concentration of 150 mg kg-1 day-1 through drinking water for 21 days. corticosterone (4-pregnen-11b-diol-3 20-dione 21-hemisuccinate; Sigma) was dissolved in vehicle (0.45% hydroxypropyl-β-cyclodextrin, Sigma). corticosterone (35 μg/mL, equivalent to 5 mg kg-1 day-1) was delivered ad libitum in the drinking water. The dose and duration of corticosterone treatment in mice were selected based on earlier studies9 (link),25 (link), where the above dose and duration of treatment with CORT induced anxiety and depressive-like behaviors in mice. The fluid intake was monitored throughout the treatment period and the corticosterone dose was adjusted accordingly. Primary cortical neurons were treated with serotonin (5-HT; Sigma) at a concentration of 5-100 μM at DIV 5-7. In glucocorticoid receptor inhibitor studies, cells were treated with RU486 (1 μM; Sigma) 30 min before 48-h corticosterone (1 μM; Sigma) treatment. Neurons were treated with BDNF (prospec) at 100ng/ml for indicated time period.
8
Calcium Signaling Modulation in Cell Experiments
9
Serotonin Transport and Toll-like Receptor Signaling
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The following drugs and substances were used (abbreviations and suppliers in parentheses): serotonin (5-HT) from Sigma-Aldrich (St. Louis, MO). [3H]-5-HT (specific activity 28 Ci/mmol) was from PerkinElmer (Boston, MA). Goat polyclonal antibody anti-human SERT (ab130130), goat polyclonal anti-human TLR10 (ab53631), and rabbit monoclonal anti-human TLR2 (ab108998) were supplied by Abcam (Cambridge, UK). Goat polyclonal anti-human actin antibody (SC-1616r) and secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA), and Pepinh-MYD (an inhibitory peptide of MyD88) was from InvivoGen (San Diego, CA). All generic reagents were purchased from Sigma-Aldrich and Roche Applied Sciences (Sant Cugat del Vallés, Barcelona, Spain).
10
Immunohistochemical Analysis of Neurotransmitter Receptors
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The following drugs and chemicals were used: Serotonin (5-HT) (Sigma-Aldrich, Merck, Saint Louis, MO, USA), Acetylcholine (Sigma-Aldrich, Merck, Saint Louis, MO, USA), mouse monoclonal antibodies SR-2A (A-4): sc-166775 or SR-2B (C-6): sc-376878 (Santa Cruz Biot., CA, USA), buffer for rinsing (50 mM TRIS, pH 7.6, 150 mM NaCl2, 0.05% Tween-20, TTBS), Biotin Blocking Kit, cat. No. BBK 120 (ScyTek Lab., Inc., Logan, UT USA); 3,3′-diaminobenzidine tetrahydrochlorid (DAB, cat. No. ACV500, Scy Tek, Lab., Inc., Logan, UT, USA), alcohol solutions (70%, 80%, 96%, 100%), xylene. All the ingredients for Krebs solution (NaCl, KCl, CaCl2, MgCl2, NaH2PO4, NaHCO3 and glucose) were obtained from Merck (Darmstadt, Germany).
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