d j 6 0 1h 8 29 d j 9 0 1h 8 72 d j 9 0 1h 13 c nmr cdcl 3 u03b4 19 5 23 4 26 5 27 4 28 0 39 1 45 9 51 8 53 2 55 4 60 2 63 8 112 5 124 2 125 0 126 5 126 7 127 6 128 7 129 8 141 4 156 3 161 2 174 4 for all receptor binding assays male sprague dawley rats rattus norvegicus albinus weighing 180 200 g were killed by decapitation and the brains rapidly removed and dissected tissues were stored at u2212 80 u00b0 c for subsequent use and homogenized on a polytron pt 10 homogenizer membrane suspensions were centrifuged on a beckman j2 hs instrument the cerebral cortex was homogenized in 10 volumes of ice cold tris buffer 50 mm tris hcl ph 7 7 at 25 u00b0 c and centrifuged at 28000 g for 15 min the membrane pellet was washed twice by resuspension and centrifugation after the second wash the resuspended pellet was incubated at 37 u00b0 c for 10 min membranes were then collected by centrifugation and the final pellet was resuspended in 50 mm tris hcl 5 mm mgso 4 and 0 5 mm edta buffer ph 7 4 at 37 u00b0 c fractions of 100 u03bcl of the final membrane suspension about 1 mg of protein were incubated at 37 u00b0 c for 15 min with 0 6 nm 3 8 oh dpat 133 ci mmol in the presence or absence of the competing drug in a final volume of 1 1 ml of assay buffer 50 mm tris hcl 10 nm clonidine 30 nm prazosin ph 7 4 at 37.