Be Aware Of SERCA Issues And also How You Can Locate It

Hypoxia was induced by lowering the O2 concentration in the inspired air down to a level of 8�C10% for 10 min. Hypercapnia was induced by titrating CO2 into the respiratory mixture up to a level of 8�C10% for 10 min. Muscimol (GABAA agonist; Sigma Chemicals Co., St Louis, MO, USA; 2 mm in sterile saline, pH 7.4) or NMDA (Sigma Chemicals Co.; 0.1 mm in sterile saline, Luminespib pH 7.4) was injected bilaterally (50 nl in each side) using a 1 ��l Hamilton syringe connected by polyethylene tubing (PE-10) to an injection needle (33 gauge) 1.5 mm longer than the guide cannulae (22 gauge) implanted in the brain (conscious rats). At the end of the experiments, rats were deeply anaesthetized with pentobarbital, and a 2% solution of Evans Blue was injected into the RTN/pFRG (50 nl). Saline followed by 10% buffered formalin (pH 7.4) was perfused through the heart. The brains were removed, fixed in 10% formalin for at least 2 days, frozen, cut coronally into 40 ��m sections and stained with Giemsa. The sections were analysed using a Zeiss Axioskop 2 microscope (Oberkochen, Germany) to confirm the location of injections into the RTN. Section SERCA alignment between brains was done relative to a reference section. To align sections around the RTN/pFRG level, the most caudal section containing an identifiable cluster of facial motor neurons was identified in each brain and assigned the level 11.6 mm caudal to bregma (bregma ?11.6 mm) according to the atlas of Paxinos & Watson (1998). Levels rostral or caudal to this reference section were determined by adding a distance corresponding to the interval between sections multiplied by the number of intervening sections. Statistical analysis was done with Sigma Stat Version Selleckchem Volasertib 3.0 (Jandel Corp., Point Richmond, CA, USA). Data are reported as means �� SEM. Student's paired t test and one-way parametric ANOVA followed by the Newman�CKeuls multiple comparisons test were used as appropriate. Significance was set at P