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All rats were used for experiments during the sixth to seventh week post-surgery. Blood pressure was measured at the beginning of the sixth <a href=""></a> week post-surgery, using a tail-cuff method. Values obtained were 137.9 �� 2.3 mmHg and 208.4 �� 3.1 mmHg in sham and hypertensive rats, respectively (P < 0.0001). Preautonomic RVLM-projecting PVN neurones were identified by injecting rhodamine beads unilaterally into the brainstem region containing the RVLM as previously described (Stern, 2001; Li et al. 2003). Rats were anaesthetized (ketamine�Cxylazine mixture, 90 and 50 mg kg?1, respectively, i.p.) and a stereotaxic apparatus was used to pressure inject 200 nl of rhodamine-labelled microspheres (Lumaflor, Naples, FL, USA) into the RVLM (starting from Bregma: 12 mm caudal along the lamina, 2 mm medial lateral, and 8 mm ventral). <a href="">Birinapant</a> In general, RVLM injection sites were contained within the caudal pole of the facial nucleus to ?1 mm more caudal, and were ventrally located with respect to the nucleus ambiguous. The location of the tracer was verified histologically (Stern, 2001; Li et al. 2003). As previously reported, retrogradely labelled neurones were found in the ventromedial (VM), dorsal cap (DC) and posterior (PaPo) PVN subnuclei (Armstrong et al. 1980; Swanson & Sawchenko, 1983; Stern, 2001; Sonner & Stern, 2007). Two to three days after the retrograde injection, rats were deeply anaesthetized with nembutal (50 mg kg?1, i.p.), and perfused through the heart with a cold sucrose solution (containing in mm: 200 sucrose, 2.5 KCl, 3 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 d-glucose, 0.4 ascorbic acid, 1 CaCl2 and 2 pyruvic acid (290�C310 mosmol l?1). Rats were then quickly decapitated, the brains were dissected out, and coronal slices cut (300 ��m thick) using a vibroslicer (D.S.K. Microslicer, Ted Pella, Redding, CA, USA). An oxygenated ice cold artificial cerebrospinal fluid (ACSF) was used during slicing (containing in mm: 119 NaCl, 2.5 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 d-glucose, 0.4 ascorbic acid, 2 CaCl2 and 2 pyruvic acid; pH 7.4; 290�C310 mosmol l?1). Slices were placed in a holding chamber containing ACSF and kept at room temperature until used (see Stern, 2001 for details). <a href="">Selleckchem Neratinib</a> Once in the recoding chamber, slices were bathed with solutions (?3.0 ml min?1) that were continuously bubbled with 95% O2�C5% CO2 and maintained at room temperature (?22�C24��C). Patch pipettes (4�C7 M��) composed of thin walled (1.5 mm outer diameter, 1.17 mm inner diameter) borosilicate glass (GC150T-7.5, Clark, Reading, UK), were pulled on a horizontal electrode puller (P-97, Sutter Instrument Co., Novato, CA, USA). The internal solution contained (mm): 140 potassium gluconate, 0.2 EGTA, 10 Hepes, 10 KCl, 0.9 MgCl2, 4 MgATP, 0.3 NaGTP and 20 sodium phosphocreatine; pH 7.2�C7.3.
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