, 2011). Injections of graded concentrations of NaCl (0.3, 0.9, and 2.1 Osm/l) were delivered through an internal carotid artery (ICA) catheter in a volume of 300 μl over a period of 10–15 s. For microdialysis, microdialysis probes Gemcitabine molecular weight were stereotaxically implanted with the U-shaped tip located within or adjacent to the right SON, as previously described (Ludwig et al., 2002): 1.0 mm posterior to bregma, 1.7 mm

lateral to midline, 9.3 mm below the surface of the skull. After an equilibration period of at least 1 hr, consecutive 30 min dialysis samples were collected at a flow rate of 3 μl/min. After two 30 min baseline periods, rats were stimulated osmotically as described above, and a further two consecutive dialysate samples were collected, frozen, and stored at −20°C until assay for VP. The VP content in the microdialysates was measured by a highly sensitive and selective radioimmunoassay (Landgraf et al., 1995). Rats were anesthetized with pentobarbital (50 mg kg−1) and perfused transcardially in 4% paraformaldehyde in 0.01 M PBS. Brains were then removed, and coronal slices (30 μm) containing the PVN were cut and incubated with one or a combination of the following primary antibodies: rabbit (1:100; Millipore) or goat (1:50; Santa Cruz Biotechnology) anti-V1a receptor; goat anti-CTB (1:2500; List Biological Laboratories); rabbit anti-TRPM4 (1:2,000;

kindly PD-1/PD-L1 inhibition donated by Dr. Teruyama, LHSU); rabbit anti-MAP2 (1:500; Sigma-Aldrich); and mouse anti-DBH (1:20,000; Millipore). Incubation in primary antibodies was followed by specific fluorescently labeled secondary antibodies (1:250; Jackson ImmunoResearch Laboratories) for 4 hr. Slices were then

ADAMTS5 rinsed and visualized using confocal microscopy (Carl Zeiss MicroImaging; 63× oil immersion, zoomed ×2; single optical plane = 0.5 μm thick) (Biancardi et al., 2010). Single-cell RT-PCR was carried out as previously described with minor modification (Sonner et al., 2011). The cytoplasm of the patched neuron, taking care not to contain the nucleus, was pulled into a patch pipette containing 2 μl DEPC-treated water and then mixed with 1 μl of RNase inhibitor (Applied Biosystems). A nested approach was used to quantify V1a receptor mRNA. The primers used included first-nested PCR (5′-CGAGGTGAACAATGGCACTAAAAC-3′ and 5′-TGTGATGGAAGGGTTTTCTGAATC-3′), second-nested PCR (5′-TCATCTGCTACCACATCTGGCG-3′ and 5′-GTGTAACCAAAAGCCCCTTATGAAAG-3′), primers for TRPM4 (5′-CCTGCAGGCCCAGGTAGAGA-3′ and 5′-TTCAGCAGAGCGTCCATGAG-3′), and GAPDH primers (5′-TTCAACGGCACAGTCAAGG-3′ and 5′-TGGTTCACACCCATCACAAA-3′). All primers were synthesized by Integrated DNA Technologies. Final PCR products were electrophoresed on a 2% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA [pH 8]) containing 0.