Samples were centrifuged at 4 °C, 3000 rpm for 20 min Plasma vas

Samples were centrifuged at 4 °C, 3000 rpm for 20 min. Plasma vasopressin levels were measured by specific radioimmunoassay after previous ABT-737 molecular weight extraction from plasma using acetone and petroleum ether (Glick and Kagan, 1979 and Elias et al., 1997). The recovery rates were greater than 87%. The assay sensitivity and intra- and inter-assay coefficients of variation were 0.9 pg/mL, 4.6 and 18.6%, respectively. All samples from a single

experiment were assayed in duplicate in the same assay. Carbachol (Sigma, St Louis, MO, USA) and CoCl2 (Sigma) were dissolved in artificial cerebrospinal fluid (aCSF), with the following composition: 100 mM NaCl, 2 mM Na3PO4, 2.5 mM KCl, 1.0 mM MgCl2, 27 mM NaHCO3 and 2.5 mM CaCl2 (pH 7.4). Tribomoethanol (Sigma) and urethane (Sigma) were dissolved in saline (0.9% NaCl). Flunixine meglumine (Banamine®, Schering Plough, Brazil) and poly-antibiotic preparation of streptomycins and penicillins (Pentabiotico®, Fort Dodge, Brazil) were used as provided. On the day of the experiment, animals were transported to the experimental room and were allowed 60 min period to adapt to the experimental room conditions, such as sound and illumination, before starting arterial

Fluorouracil chemical structure pressure and HR recording. The experimental room was acoustically isolated and a constant background noise was generated by an air exhauster to minimize sound interference within the experimental room. This experiment aimed to study the

effect of carbachol microinjection into the BST of unanesthetized rats on plasma vasopressin levels. For this, two different groups of animals received vehicle (aCSF, 100 nL, n = 6) or carbachol (1 nmol/100 nL, n = 6) injected into the BST (Alves et al., 2007). Five minutes after BST treatment, animals were decapitated and blood samples were collected to determine of plasma vasopressin levels. These experiments aimed to study the involvement of the SON in cardiovascular responses to carbachol microinjection into the BST of unanesthetized rats. For this, animals were divided into two groups, ipsilateral and contralateral SON groups. In the ipsilateral SON group, rats had cannulas implanted unilaterally in the BST and in the ipsilateral SON, in relation to BST cannula, and were Cyclic nucleotide phosphodiesterase subdivided into vehicle (aCSF, 100 nL, n = 7) and CoCl2 (1 mM/100 nL, n = 7) groups (Busnardo et al., 2007, Crestani et al., 2009a, Crestani et al., 2009b and Scopinho et al., 2008). In the contralateral SON group, rats had cannulas implanted unilaterally in the BST and in the contralateral SON and were subdivided into vehicle (aCSF, 100 nL, n = 6) and CoCl2 (1 mM/100 nL, n = 6) groups (Alves et al., 2007, Busnardo et al., 2007, Crestani et al., 2009a and Crestani et al., 2009b). Carbachol (1 nmol/100 nL) was microinjected into the BST on the first day and again 24 h later, at 10 min after aCSF or CoCl2 microinjection into the SON (Alves et al., 2007).

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