Substance p sp

The pharmacological cocktail used to synaptically isolate pacemaker neurons contained 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 μM DL-2-amino-5-phosphonovaleric acid (AP5), 1 μM strychnine, and 10 μM bicuculline (all from Sigma).
Substance P (SP, Sigma) and the μ opioid receptor agonist D-Ala²-N-Me-Phe⁴-Glycol⁵-enkephalin (DAMGO, Sigma) were dissolved in aCSF and bath-applied at their final concentration of 0.5 μM and 0.25 μM, respectively. A single modulatory drug was applied per preparation in order to avoid cross effects. Preparations were exposed to a drug for a 15 min period and its effects on respiratory rhythm cycle frequency were analyzed at the end of this period using Clampfit software (Molecular Devices). The reversal of any observed effects was tested for each drug after a 30 min period wash-out during which fresh aCSF was superfused in the recording chamber.
Values are given as mean ± SEM. Student's t tests with a Mann-Whitney Rank Sum test or ANOVA tests were used to assess differences (taken to be significant at P < 0.05) between values in the presence of the drugs and controls.

To investigate the phenotypical changes of microglia, MG d14 were pre-treated with 10 ng/mL GMCSF (R&D Systems, Minneapolis, MN, USA) for 5 days and then incubated with the following cytokines for 3 days: 10 ng/mL GMCSF (R&D systems, Minneapolis, MN, USA), 100 nM Substance P (SP, Sigma, St. Louis, MO, USA), 10 ng/mL rat IL-4 (R&D systems, Minneapolis, MN, USA), 10 ng/mL rat IL-10 (R&D systems, Minneapolis, MN, USA), 10 ng/mL TNF-α (Sigma, St. Louis, MO, USA), and 20 ng/mL IFN-γ (R&D systems, Minneapolis, MN, USA).

Injections were given in the loose connective tissue outside the Achilles tendon, i.e. in the paratenon region, of the right leg. The injections were given directly after each of the exercise regimens. The substances injected were Captopril (C) (c4042; Sigma), DL-Thiorphan (Th) (T 6031; Sigma), substance P (SP) (S6883; Sigma) and NaCl. Captopril is an angiotensin-converting enzyme inhibitor (ACE inhibitor) and DL-Thiorphan inhibits the activity of neutral endopeptidase (NEP). NEP is also called enkephalinase and neprilysin or common acute lymphoblastic leukemia antigen or CD10 [29 (link)].
Injections were performed as follows: a) Substance P (10^-8 μmol/ml) and Captopril (30 μmol/kg) both in distilled water (volume: 1 ml) + DL-Thiorphan (500 μg/ml; 0.02 ml) (5 animals), b) Captopril (30 μmol/kg, dissolved in distilled water, volume 1 ml) + DL-Thiorphan (500 μg/ml, 0.02 ml) (6 animals), c) NaCl (volume: 1 ml) (5 animals). The groups are further referred to as the SP + C + Th group, the C + Th group and the NaCl group.