Resulting data points were fitted to a dose–response curve. The dose of 45 nmol/50 nL was used in the following protocols and 50 nL of ACSF was microinjected as vehicle control. Number of rats used, n = 12. These doses were established from data in the literature ( Pizzirusso et al., 1998). The third group of animals was used to evaluate the involvement of muscarinic receptors in the cardiovascular response to the injection of Ach into the vlPAG. Different doses of the nonselective muscarinic receptor antagonist atropine (1,
3 or 9 nmol/50 nL) were microinjected into the vlPAG 10 min before microinjection of Ach. Each animal received only one dose of atropine. Number of rats used, n = 16. These doses were established from data in the literature ( Crippa et al., 1999). In the last part of the study, we determined whether the cardiovascular response was due to a central effect of Ach. Animals received this website intravenously the same dose of atropine as injected into the vlPAG (9 nmol) 10 min prior to the injection of 45 nmol of Ach into that area. Number of rats used, n = 6. At the end of the experiments, 50 nL of 1% Evan’s blue dye was injected into the vlPAG or the dPAG as a marker of the injection site. Animals were submitted to intracardiac perfusion with 0.9% NaCl followed by 10% formalin. Brains were removed SGI-1776 research buy and post-fixed for 48 h at 4 °C and serial 40 μm-thick sections were cut using a cryostat (CM1900,
Leica, Germany). Brain sections were stained with 1% neutral red for light microscopy analysis. The actual location of microinjection sites in the area was determined after the analysis of serial sections and represented according to the rat brain atlas of Paxinos and Watson (1997). Nonlinear regression analysis
was used to compare MAP and HR results from different Ach doses microinjected into the vlPAG or the dPAG. Baseline MAP and HR values were compared using the paired Student’s t test (before treatment vs. after treatment). Percentages of response inhibition by vlPAG pretreatment with muscarinic antagonists were analysed utilizing nonlinear regression Clomifene analysis. We used Prism software (GraphPad, USA) to perform statistical analysis. *P < 0.05 was assumed to be statistically significant. The authors would like to thank Ms. Ivanilda A.C. Fortunato, Idália I.B. Aguiar and Simone S. Guilhaume for technical support. Cristiane Busnardo (Fapesp proc. 2009/05308-8) is a post-doctoral fellow in the Department of Pharmacology of the School of Medicine of Ribeirão Preto-USP. Milena Vieira Deolindo (Fapesp proc. 07/50166-1) is a PhD student enrolled in the Graduation Program on Pharmacology of the School of Medicine of Ribeirão Preto-USP. "
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